Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant

ABSTRACT

The present invention relates generally to adjuvants, and in particular to methods and products utilizing a synergistic combination of immunostimulatory oligonucleotides having at least one unmethylated CpG dinucleotide (CpG ODN) and a non-nucleic acid adjuvant. Such combinations of adjuvants may be used with an antigen or alone. The present invention also relates to methods and products utilizing immunostimulatory oligonucleotides having at least one unmethylated CpG dinucleotide (CpG ODN) for induction of cellular immunity in infants.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 10/023,909, filed on Dec. 18, 2001, and now pending, which is adivisional of U.S. patent application Ser. No. 09/325,193, filed on Jun.3, 1999, and now Issued as U.S. Pat. No. 6,404,705, which is acontinuation in part of U.S. patent application Ser. No. 09/154,614filed on Sep. 16, 1998, abandoned, which is a National Stage filing ofPCT/US98/04703, filed on Mar. 10, 1998, claiming priority to U.S.Provisional Patent Application 60/040,376, filed Mar. 10, 1997, nowabandoned, the entire contents each of which are incorporated byrefernce.

FIELD OF THE INVENTION

[0002] The present invention relates generally to adjuvants, and inparticular to methods and products utilizing a synergistic combinationof oligonucleotides having at least one unmethylated CpG dinucleotide(CpG ODN) and a non-nucleic acid adjuvant.

BACKGROUND OF THE INVENTION

[0003] Bacterial DNA, but not vertebrate DNA, has directimmunostimulatory effects on peripheral blood mononuclear cells (PBMC)in vitro (Krieg et al., 1995). This lymphocyte activation is due tounmethylated CpG dinucleotides, which are present at the expectedfrequency in bacterial DNA ({fraction (1/16)}), but areunder-represented (CpG suppression, {fraction (1/50)} to {fraction(1/60)}) and methylated in vertebrate DNA. Activation may also betriggered by addition of synthetic oligodeoxynucleotides (ODN) thatcontain an unmethylated CpG dinucleotide in a particular sequencecontext. It appears likely that the rapid immune activation in responseto CpG DNA may have evolved as one component of the innate immunedefense mechanisms that recognize structural patterns specific tomicrobial molecules.

[0004] CpG DNA induces proliferation of almost all (>95%) B cells andincreases immunoglobulin (Ig) secretion. This B cell activation by CpGDNA is T cell independent and antigen non-specific. However, B cellactivation by low concentrations of CpG DNA has strong synergy withsignals delivered through the B cell antigen receptor for both B cellproliferation and Ig secretion (Krieg et al., 1995). This strong synergybetween the B cell signaling pathways triggered through the B cellantigen receptor and by CpG DNA promotes antigen specific immuneresponses. In addition to its direct effects on B cells, CpG DNA alsodirectly activates monocytes, macrophages, and dendritic cells tosecrete a variety of cytokines, including high levels of IL-12 (Klinmanet al., 1996; Halpern et al., 1996; Cowdery et al., 1996). Thesecytokines stimulate natural killer (NK) cells to secretegamma-interferon (IFN- −) and have increased lytic activity (Klinman etal., 1996, supra; Cowdery et al., 1996, supra; Yamamoto et al., 1992;Ballas et al., 1996). Overall, CpG DNA induces a Th1 like pattern ofcytokine production dominated by IL-12 and IFN-γ with little secretionof Th2 cytokines (Klinman et al., 1996).

[0005] Hepatitis B virus (HBV) poses a serious world-wide healthproblem. The current HBV vaccines are subunit vaccines containingparticles of HBV envelope protein(s) which include several B and T cellepitopes known collectively as HBV surface antigen (HBsAg). The HBsAgparticles may be purified from the plasma of chronically infectedindividuals or more commonly are produced as recombinant proteins. Thesevaccines induce antibodies against HBsAg (anti-HBs), which conferprotection if present in titers of at least 10 milli-International Unitsper milliliter (mIU/ml) (Ellis, 1993). The current subunit vaccines whchcontain alum (a Th2 adjuvant), are safe and generally efficacious. They,however, fail to meet all current vaccination needs. For example, earlyvaccination of infants born to chronically infected mothers, as well asothers in endemic areas, drastically reduces the rate of infection, buta significant proportion of these babies will still become chronicallyinfected themselves (Lee et al., 1989; Chen et al., 1996). This couldpossibly be reduced if high titers of anti-HBs antibodies could beinduced earlier and if there were HBV-specific CTL. In addition, thereare certain individuals who fail to respond (non-responders) or do notattain protective levels of immunity (hypo-responders). Finally, thereis an urgent need for an effective treatment for the estimated 350million chronic carriers of HBV and a therapeutic vaccine could meetthis need.

SUMMARY OF THE INVENTION

[0006] The present invention relates to methods and products forinducing an immune response. The invention is useful in one aspect as amethod of inducing an antigen specific immune response in a subject. Themethod includes the steps of administering to the subject in order toinduce an antigen specific immune response an antigen and a combinationof adjuvants, wherein the combination of adjuvants includes at least oneoligonucleotide containing at least one unmethylated CpG dinucleotideand at least one non-nucleic acid adjuvant, and wherein the combinationof adjuvants is administered in an effective amount for inducing asynergistic adjuvant response. In one embodiment the subject is aninfant.

[0007] The CpG oligonucleotide and the non-nucleic acid adjuvant may beadministered with any or all of the administrations of antigen. Forinstance the combination of adjuvants may be administered with a primingdose of antigen. In another embodiment the combination of adjuvants isadministered with a boost dose of antigen. In some embodiments thesubject is administered a priming dose of antigen and oligonucleotidecontaining at least one unmethylated CpG dinucleotide before the boostdose. In yet other embodiments the subject is administered a boost doseof antigen and oligonucleotide containing at least one unmethylated CpGdinucleotide after the priming dose.

[0008] The antigen may be any type of antigen known in the art. Forexample, the antigen may be selected from the group consisting ofpeptides, polypeptides, cells, cell extracts, polysaccharides,polysaccharide conjugates, lipids, glycolipids and carbohydrates.Antigens may be given in a crude, purified or recombinant form andpolypeptide/peptide antigens, including peptide mimics ofpolysaccharides, may also be encoded within nucleic acids. Antigens maybe derived from an infectious pathogen such as a virus, bacterium,fungus or parasite, or the antigen may be a tumor antigen, or theantigen may be an allergen.

[0009] According to another aspect of the invention a method of inducinga Th1 immune response in a subject is provided. The method includes thestep of administering to the subject in order to induce a Th1 immuneresponse a combination of adjuvants, wherein the combination ofadjuvants includes at least one oligonucleotide containing at least oneunmethylated CpG dinucleotide and at least one non-nucleic acidadjuvant, and wherein the combination of adjuvants is administered in aneffective amount for inducing a Th1 immune response. In one embodimentthe combination of adjuvants is administered simultaneously. In anotherembodiment the combination of adjuvants is administered sequentially. Insome embodiments the combination of adjuvants is administered in aneffective amount for inducing a synergistic Th1 immune response. Inanother aspect, the same method is performed but the subject is aninfant and the Th1 response can be induced using CpG DNA alone, or CpGDNA in combination with a non-nucleic acid adjuvant at the same ordifferent site, at the same or different time.

[0010] The invention in other aspects is a composition of a synergisticcombination of adjuvants. The composition includes an effective amountfor inducing a synergistic adjuvant response of a combination ofadjuvants, wherein the combination of adjuvants includes at least oneoligonucleotide containing at least one unmethylated CpG dinucleotideand at least one non-nucleic acid adjuvant. The composition may alsoinclude at least one antigen, which may be selected from the groupconsisting of peptides, polypeptides, cells, cell extracts,polysaccharides, polysaccharide conjugates, lipids, glycolipids andcarbohydrates. Antigens may be given in a crude, purified or recombinantform and polypeptide/peptide antigens, including peptide mimics ofpolysaccharides, may also be encoded within nucleic acids. Antigens maybe derived from an infectious pathogen such as a virus, bacterium,fungus or parasite, or the antigen may be a tumor antigen, or theantigen may be an allergen.

[0011] According to other aspects the invention includes a method forimmunizing an infant. The method involves the step of administering toan infant an antigen and an oligonucleotide containing at least oneunmethylated CpG dinucleotide and at least one non-nucleic acid adjuvantin an effective amount for inducing cell mediated immunity or Th1-likeresponses in the infant. The method may also involve the step ofadministering at least one non-nucleic acid adjuvant.

[0012] The CpG oligonucleotide may be administered with any or all ofthe administrations of antigen. For instance the CpG oligonucleotide orthe combination of adjuvants may be administered with a priming dose ofantigen. In another embodiment the CpG oligonucleotide or thecombination of adjuvants is administered with a boost dose of antigen.In some embodiments the subject is administered a priming dose ofantigen and oligonucleotide containing at least one unmethylated CpGdinucleotide before the boost dose. In yet other embodiments the subjectis administered a boost dose of antigen and oligonucleotide containingat least one unmethylated CpG dinucleotide after the priming dose.

[0013] The invention in other aspects includes a method of inducing astronger Th1 immune response in a subject being treated with anon-nucleic acid adjuvant. The method involves the steps ofadministering to a subject receiving an antigen and at least onenon-nucleic acid adjuvant and at least one oligonucleotide containing atleast one unmethylated CpG dinucleotide in order to induce a strongerTh1 immune response than either the adjuvant or oligonucleotide producesalone.

[0014] The invention in other aspects include a method of inducing anon-antigen-specific Th1-type immune response, including Th1 cytokinessuch as IL-12 and IFN-, for temporary protection against variouspathogens including viruses, bacteria, parasites and fungi. The methodinvolves the steps of administering to a subject at least onenon-nucleic acid adjuvant and at least one oligonucleotide containing atleast one unmethylated CpG dinucleotide in order to induce a Th1 innateimmune response. For longer term protection, these adjuvants may beadministered more than once. In another embodiment, CpG DNA may be usedalone at one or more of the administrations.

[0015] In each of the above described embodiments a CpG oligonucleotideis used as an adjuvant. The oligonucleotide in one embodiment containsat least one unmethylated CpG dinucleotide having a sequence includingat least the following formula:

5′X₁X₂CGX₃X₄3′

[0016] wherein C and G are unmethylated, wherein X₁X₂ and X₃X4 arenucleotides. In one embodiment the 5′X₁ X₂CGX₃ X₄ 3′ sequence is anon-palindromic sequence.

[0017] The oligonucleotide may be modified. For instance, in someembodiments at least one nucleotide has a phosphate backbonemodification. The phosphate backbone modification may be aphosphorothioate or phosphorodithioate modification. In some embodimentsthe phosphate backbone modification occurs on the 5′ side of theoligonucleotide or the 3′ side of the oligonucleotide.

[0018] The oligonucleotide may be any size. Preferably theoligonucleotide has 8 to 100 nucleotides. In other embodiments theoligonucleotide is 8 to 40 nucleotides in length.

[0019] In some embodiments X₁X₂ are nucleotides selected from the groupconsisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT,and TpG; and X₃X₄ are nucleotides selected from the group consisting of:TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA.Preferably X₁X₂ are GpA or GpT and X₃X₄ are TpT. In other preferredembodiments X₁ or X₂ or both are purines and X₃ or X₄ or both arepyrimidines or X₁X₂ are GpA and X₃ or X₄ or both are pyrimidines. In oneembodiment X₂ is a T and X₃ is a pyrimidine. The oligonucleotide may beisolated or synthetic.

[0020] The invention also includes the use of a non-nucleic acidadjuvant in some aspects. The non-nucleic acid adjuvant in someembodiments is an adjuvant that creates a depo effect, an immunestimulating adjuvant, or an adjuvant that creates a depo effect andstimulates the immune system. Preferably the adjuvant that creates adepo effect is selected from the group consisting of alum (e.g.,aluminum hydroxide, aluminum phosphate) emulsion based formulationsincluding mineral oil, non-mineral oil, water-in-oil or oil-in-wateremulsions, such as the Seppic ISA series of Montanide adjuvants; MF-59;and PROVAX. In some embodiments the immune stimulating adjuvant isselected from the group consiting of saponins purified from the bark ofthe Q. saponaria tree, such as QS21;poly[di(carboxylatophenoxy)phosphazene (PCPP) derivatives oflipopolysaccharides such as monophosphorlyl lipid (MPL), muramyldipeptide (MDP) and threonyl muramyl dipeptide (tMDP); OM-174; andLeishmania elongation factor. In one embodiment the adjuvant thatcreates a depo effect and stimulates the immune system is selected fromthe group consiting of ISCOMS; SB-AS2; SB-AS4; non-ionic blockcopolymers that form micelles such as CRL 1005; and Syntex AdjuvantFormulation.

[0021] Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 has two graphs illustrating humoral and cytotoxicT-lymphocyte (CTL) responses in adult BALB/c mice immunized with 1 μgrecombinant HBsAg protein alone, adsorbed onto alum (25 mg Al³⁺/mgHBsAg), with 100 μg of immunostimulatory CpG ODN 1826, or with both alumand CpG ODN. Left panel: Each point represents the group mean (n=10) fortiters of anti-HBs (total IgG) as determined in triplicate by end-pointdilution ELISA assay. End-point titers were defined as the highestplasma dilution that resulted in an absorbance value (OD 450) two timesgreater than that of control non-immune plasma with a cut-off value of0.05. Right panel: Each point represents the mean % specific lysis atthe indicated effector: target (E:T) cell ratio in a chromium releaseassay with HBsAg-expressing cells as targets.

[0023]FIG. 2 is a graph illustrating humoral responses in adult BALB/cmice immunized with 1 μg recombinant HBsAg protein, with or withoutalum, and with 0, 0.1, 1, 10, 100 or 500 μg of CpG ODN 1826 added. Eachpoint represents the group mean (n=10) for anti-HBs titers (total IgG)as determined by end-point dilution ELISA assay.

[0024]FIG. 3 is a graph illustrating humoral responses in adult BALB/cmice immunized with 1 μg recombinant HBsAg protein, with or withoutalum, and with one of several different oligonucleotides (ODN, 10 μg).The ODN were made with a natural phosphodiester backbone (O), syntheticphosphorothioate backbone (S) or a chimeric of phosphodiester centerregions and phosphorothioate ends (SOS). Most of the ODN contained 1-3CpG motifs but some of the ODN were non-CpG controls (1911, 1982, 2041).Each point represents the group mean (n=5) for anti-HBs titers (totalIgG) as determined by end-point dilution ELISA assay.

[0025]FIG. 4 is a graph of CTL responses in adult BALB/c mice immunizedwith 1 μg recombinant HBsAg protein with alum (25 mg Al³⁺/mg HBs/Ag),with 10 μg of CpG ODN 1826, or with both alum and CpG ODN. Some animalswere boosted with the same or a different formulation after 8 weeks.Each point represents the group mean (n=5) for % specific lysis ofHBsAg-expressing target cell at various effector:target (E:T) cellratios.

[0026]FIG. 5 is a graph of humoral responses in BALB/c mice immunizedwith HBsAg (1 μg) without adjuvant or with various adjuvants alone or incombination. The adjuvants were: alum (25 mg Al³⁺/mg HBs/Ag), with CpGDNA (10 μg CpG ODN 1826), monophosphoryl lipid A (MPL, 50 μg) andFreund's complete adjuvant (mixed 1:1 v/v with HBsAg solution). Eachpoint represents the group mean (n=10) for anti-HBS titers (total IgG)as determined by end-point dilution ELISA assay 4 weeks afterimmunization.

[0027]FIG. 6 is a bar graph depicting the amount of total IgG (end-pointELISA titer) produced at 4 weeks in BALB/c mice immunized with 1 μg ofHBsAg with or without CpG and/or IFA (mineral oil mixed 1:1 v/v) or CFA(complete Freund's adjuvant mixed 1:1 v/v). The numbers above each barindicate the IgG2a:IgG1 ratio, with a number in excess of 1 indicating amore Th1-like response.

[0028]FIG. 7 is a bar graph depicting the amount of total IgG producedat 4 weeks in BALB/c mice immunized with 1 μg of HBsAg with or withoutCpG and/or MPL (monophosphoryl lipid A, 50 μg) or alum. The numbersabove each bar indicate the IgG2a:IgG1 ratio, with a number in excess of1 indicating a more Th1-like response.

[0029]FIG. 8 is a graph of the percent of young BALB/c mice thatseroconverted (end-point dilution titer >100) after immunization at <1,3, 7 or 14 days of age. Mice were immunized with 10 μg HBsAg-expressingDNA vaccine (pCMV-S), or with recombinant HBsAg (1 μg) with alum (25 mgAl3+/mg HBsAg), CpG ODN 1826 (10 μg) or both alum and CpG ODN. Eachpoint represents the proportion of mice responding, the numbers abovethe bars show the number of responding over the total number immunized.

[0030]FIG. 9 has two graphs illustrating humoral and cytotoxicT-lymphocyte (CTL) responses in BALB/c mice immunized at 7 days of agewith a DNA vaccine (1 μg pCMV-S), or with 1 μg recombinant HBsAg proteinalone, adsorbed onto alum (25 mg Al³⁺/mg HBsAg), with 100 μg ofimmunostimulatory CpG ODN 1826, or with both alum and CpG ODN. Upperpanel: Each point represents the group mean of animals thatseroconverted (see FIG. 8 for numbers of animals) for titers of anti-HBs(total IgG) as determined in triplicate by end-point dilution ELISAassay. End-point titers were defined as the highest plasma dilution thatresulted in an absorbance value (OD 450) two times greater than that ofcontrol non-immune plasma with a cut-off value of 0.05. Lower panel.Each point represents the mean % specific lysis at the indicatedeffector: target (E:T) cell ratio in a chromium release assay withHBsAg-expressing cells as targets.

[0031]FIG. 10 is a bar graph illustrating humoral responses in neonatalBALB/c mice at 8 weeks after immunization (at 7 days of age) with 1 μgrecombinant HBsAg protein with alum (25 mg Al³⁺/mg HBsAg), with 10 μg ofCpG ODN 1826, or with both alum and CpG ODN. Each point represents thegroup mean (see FIG. 8 for numbers of animals) for anti-HBs titers (IgG1and IgG2a isotypes) as determined by end-point dilution ELISA assay.IgG1 antibodies indicate a Th2-biased response whereas IgG2a antibodiesare indicative of a Th1-type response.

[0032]FIG. 11 is a graph of humoral responses in juvenile Cynomolgusmonkeys immunized with Engerix-B vaccine (10 μg recombinant HBsAgprotein with alum, SmithKline Beecham biologicals, Rixensart, BE) orwith Engerix-B plus 500 μg of CpG ODN 1968. Each point represents thegroup mean (n=5) for anti-HBs titers in milli-International units/ml(mIU/ml). A titer of 10 mIU/ml is considered protective in humans.

[0033]FIG. 12 is a bar graph depicting titers of antibodies againstHBsAg (anti-HBs) in milliInternational Units per millilitre (mIU/ml) inorangutans immunized with 10 μg HBsAg with alum (like the HBV commercialvaccine), CpG oligonucleotides (CpG ODN 2006, 1 mg) or both alum and CpGODN. The numbers above the bars show the number of animals withseroconversion (upper numbers, >1 mIU/ml) or with seroprotection (lowernumbers, >10 mIU/ml) over the total number of animals immunized. A titerof 10 mIU/ml is considered sufficient to protect humans and great apesagainst infection.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The invention in one aspect is based on the discovery thatformulations containing combinations of immunostimulatory CpGoligonucleotides and non-nucleic acid adjuvants synergistically enhanceimmune responses to a given antigen. Different non-nucleic acidadjuvants used in combination in the prior art have different affects onimmune system activation. Some combinations of adjuvants produce anantigen-specific response that is no better than the best of theindividual components and some combinations even produce lower antigenspecific responses than with the individual adjuvants used alone. InGordon et al., for instance, when humans were immunized with C terminalrecombinant malaria circumsporozite antigen with alum alone or alum incombination with monophosphoryl lipid A (MPL), the subjects receivingalum alone developed higher antigen specific antibodies at several timepoints than subjects receiving the combination of adjuvants.

[0035] It has been discovered according to the invention that thecombination of immunostimulatory CpG oligonucleotides and alum, MPL andother adjuvants results in a synergistic immune response. Compared withthe recombinant hepatitis B surface antigen (HBsAg) protein vaccinealone, addition of alum increases the level of antibodies in miceagainst HBsAg (anti-HBs) about 7-fold whereas addition of CpG ODNincreases them 32-fold. When CpG ODN and alum are used together, a500-1000 times higher level of anti-HBs was observed, indicating astrong synergistic response. Additionally, it was found according to theinvention that immunization with HBsAg and alum resulted in a strongTh2-type response with almost all IgG being of the IgG1 isotype. CpG ODNinduced a high proportion of IgG2a, indicative of a Th1-type response,even in the presence of alum. Furthermore, it was discovered accordingto the invention that in very young mice (7 day old), immune responseswere induced by HBsAg with alum and CpG ODN but not with alum or CpG ODNalone. The antibodies produced with CpG ODN were predominantly of theIgG2a isotype, indicating a strong Th1-type response. This is remarkableconsidering the strong Th2 bias of the neonatal immune system and theknown difficulty in inducing Th1 responses at such a young age. Th1responses are preferable in some instances since they are associatedwith IgG2a antibodies that have better neutralization and opsonizationcapabilities than Th2-type antibodies. As well, Th1 responses areassociated with cytotoxic T lymphocytes (CTL) that can attack and killvirus-infected cells. Indeed, CpG ODN, alone or in combination with aluminduced good CTL activity in both adult and neonatal mice. These studiesdemonstrate that the addition of CpG ODN to protein or DNA vaccines incombination with other adjuvants is a valid new adjuvant approach toimprove efficacy.

[0036] Thus in one aspect the invention is a method of inducing anantigen specific immune response in a subject. The method includes thestep of administering to the subject in order to induce an antigenspecific immune response an antigen and a combination of adjuvants,wherein the combination of adjuvants includes at least oneoligonucleotide containing at least one unmethylated CpG dinucleotideand at least one non-nucleic acid adjuvant, and wherein the combinationof adjuvants is administered in an effective amount for inducing asynergistic adjuvant response.

[0037] The synergistic combination of adjuvants is particularly usefulas a prophylactic vaccine for the treatment of a subject at risk ofdeveloping an infection with an infectious organism or a cancer in whicha specific cancer antigen has been identified or an allergy where theallergen is known. The combination of adjuvants can also be givenwithout the antigen or allergen for shorter term protection againstinfection, allergy or cancer, and in this case repeated doses will allowlonger term protection. A “subject at risk” as used herein is a subjectwho has any risk of exposure to an infection causing pathogen or acancer or an allergen or a risk of developing cancer. For instance, asubject at risk may be a subject who is planning to travel to an areawhere a particular type of infectious agent is found or it may be asubject who through lifestyle or medical procedures is exposed to bodilyfluids which may contain infectious organisms or even any subject livingin an area that an infectious organism or an allergen has beenidentified. Subjects at risk of developing infection also includegeneral populations to which a medical agency recommends vaccinationwith a particular infectious organism antigen. If the antigen is anallergen and the subject develops allergic responses to that particularantigen and the subject is exposed to the antigen, i.e., during pollenseason, then that subject is at risk of exposure to the antigen.

[0038] There is a need for a prophylactic vaccine that can induceprotective immunity against many infectious pathogens more quickly andwith fewer doses than traditional vaccines can provide. For instance,fewer than 20% of healthy individuals attain protective levels ofanti-hepatitis B (HB) antibodies (10 mIU/ml) after a single dose ofsubunit hepatitis B Vaccine (HBV) vaccine and only 60-70% reach thislevel after two doses. Thus, three doses (usually given at 0, 1 and 6months) are required to seroconvert >90% of vaccinated individuals. Thethree dose regime is frequently not completed owing to poor patientcompliance, and in endemic areas, protective levels may not be inducedquickly enough. The methods of the invention are particularly useful asprophylactic treatments because they induce higher levels of antibodiesthan can be achieved with traditional vaccines and can be administeredas fewer total doses.

[0039] Additionally between 5 and 10% of individuals are non-respondersor hypo-responders to the subunit HBsAg vaccine. This may beMHC-restricted (Kruskall et al., 1992) and is thought to result from afailure to recognize T-helper epitopes. In certain immunocompromisedindividuals (e.g., kidney dialysis patients, alcoholics) the rate ofnon-response can approach 50%. As set forth in the Examples below, alumplus CpG ODN gave higher anti-HBs titers than alum alone in a strain ofmice which has MHC-restricted hypo-responsiveness to HBsAg, thought toresult in a failure to recognize T-helper epitopes. CpG ODN alsoovercame non-response in mice genetically incapable of providing T-helpowing to an absence of class II MHC. Similar results were obtained inorangutans at risk of becoming infected with hepatitis B. It was foundthat orangutans are hyporesponders to the classical alum-adjuvantedvaccine with less than 10% achieving seroprotection after 2 doses, butthat nearly 100% of animals responded with use of CpG oligonucleotidesalone or combined with alum. The synergistic response was evidentbecause antibody titers were much higher with CpG ODN plus alum thanwith CpG ODN alone or alum alone and were more than additive. Theseresults support the proposition that CpG ODN drives the T cellindependent activation of B cells. Thus in addition to providing a moreeffective and easier vaccination protocol the synergistic combination ofadjuvants can be used to reduce the percentage of non-responders andhypo-responders.

[0040] A subject at risk of developing a cancer is one who is who has ahigh probability of developing cancer. These subjects include, forinstance, subjects having a genetic abnormality, the presence of whichhas been demonstrated to have a correlative relation to a higherlikelihood of developing a cancer and subjects exposed to cancer causingagents such as tobacco, asbestos, or other chemical toxins, or a subjectwho has previously been treated for cancer and is in apparent remission.When a subject at risk of developing a cancer is treated with an antigenspecific for the type of cancer to which the subject is at risk ofdeveloping and an adjuvant and a CpG oligonucleotide the subject may beable to kill of the cancer cells as they develop. If a tumor begins toform in the subject, the subject will develop a specific immune responseagainst the tumor antigen.

[0041] In addition to the use of the combination of adjuvants forprophylactic treatment, the invention also encompasses the use of thecombination for the immunotherapeutic treatment of a subject having aninfection, an allergy or a cancer. A “subject having an infection” is asubject that has been exposed to an infectious pathogen and has acute orchronic detectable levels of the pathogen in the body. The combinationof adjuvants can be used with an antigen to mount an antigen specificimmune response that is capable of reducing the level of or eradicatingthe infectious pathogen. An infectious disease, as used herein, is adisease arising from the presence of a foreign microorganism in thebody.

[0042] Many types of infectious pathogens do not have any effectivetreatments and chronic presence of the pathogen can result insignificant damage. For instance, the HBV virus is itself non-pathogenicbut with chronic infection the partially developed immune responsecauses inflammatory changes that eventually leads to cirrhosis andincreased risk of hepatocellular carcinoma. An estimated one millionpeople die each year from HBV-related liver disease. Persistent HBVinfection of the liver results when acute infection fails to launch anappropriate immune response to clear the virus. Such chronic carriershave circulating HBsAg “e” soluble form of the HBV core antigen (HBeAg)without specific immunity. It is thought that the absence ofHBV-specific T-cells, including CTL may contribute to the establishmentand maintenance of the chronic carrier state. Indeed, many previouslyinfected individuals, even years after clinical and serologicalrecovery, have traces of HBV in their blood and HBV-specific CTL thatexpress activation markers indicative of recent contact with antigen(Rehermann et al., 1996). These results suggest that sterilizingimmunity may not occur after HBV infection and that chronic activationof HBV-specific CD4+ and CD8+T-cells is responsible for keeping thevirus under control.

[0043] There is currently no cure for the HBV chronic infection.Interferon is used currently but this cures only 10-20% of treatedindividuals (Niederau et al., 1996). Anti-viral drugs (e.g., lamivudine)can reduce circulating virus to undetectable levels, however thesereturn to pretreatment levels if the drug is stopped. Each of thesetypes of treatment is also expensive and has certain undesirableside-effects. Thus the synergistic combination of adjuvants whichinduces potent Th1 responses, including CTL, is useful for treating asubject having an infection such as HBV.

[0044] A “subject having an allergy” is a subject that has or is at riskof developing an allergic reaction in response to an allergen. An“allergy” refers to acquired hypersensitivity to a substance (allergen).Allergic conditions include but are not limited to eczema, allergicrhinitis or coryza, conjunctivitis, hay fever, bronchial asthma,urticaria (hives) and food allergies, and other atopic conditions.

[0045] Currently, allergic diseases are generally treated eithersymptomatically with antihistimines for example or immunotherapeuticallyby the injection of small doses of antigen followed by subsequentincreasing dosage of antigen. Symptomatic treatment offers onlytemporary relief. Immunotherapy is believed to induce tolerance to theallergen to prevent further allergic reactions. This approach, however,takes several years to be effective and is associated with the risk ofside effects such as anaphylactic response. The methods of the inventionavoid these problems.

[0046] Allergies are generally caused by IgE antibody generation againstharmless allergens. The cytokines that are induced by unmethylated CpGoligonucleotides are predominantly of a class called “Th1” whichincludes IL-12 and IFN-γ. In contrast, Th2 immune response areassociated with the production of IL-4, IL-5 and IL-10. Th1 responsesinclude both cell-mediated responses (including cytotoxic T-cells) andantibodies, whereas Th2 responses are associated only with antibodies.The antibodies with a Th1 response are of isotypes (e.g. IgG2a) thathave better neutralizing and opsonizing capabilities than those of Th2isotypes (e.g. IgE that mediates allergic responses). In general, itappears that allergic diseases are mediated by Th2 type immune responsesand protective immune responses by Th1 immune response althoughexaggerated Th1 responses may be also associated with autoimmunediseases.

[0047] Th2 cytokines, especially IL-4 and IL-5 are elevated in theairways of asthmatic subjects. These cytokines promote important aspectsof the asthmatic inflammatory response, including IgE isotype switching,eosinophil chemotaxis and activation and mast cell growth. Th1cytokines, especially IFN- and IL-12, can suppress the formation of Th2clones and production of Th2 cytokines. “Asthma” refers to a disorder ofthe respiratory system characterized by inflammation, narrowing of theairways and increased reactivity of the airways to inhaled agents.Asthma is frequently, although not exclusively associated with atopic orallergic symptoms.

[0048] Based on the ability of the CpG oligonucleotides to shift theimmune response in a subject from a Th2 (which is associated withproduction of IgE antibodies and allergy) to a Th1 response (which isprotective against allergic reactions), an effective dose of a CpGoligonucleotide can be administered to a subject to treat or prevent anallergy.

[0049] Since Th1 responses are even more potent with CpG DNA combinedwith non-nucleic acid adjuvants, the combination of adjuvants of thepresent invention will have significant therapeutic utility in thetreatment of allergic conditions such as asthma. Such combinations ofadjuvants could be used alone or in combination with allergens.

[0050] A “subject having a cancer” is a subject that has detectablecancerous cells. The cancer may be a malignant or non-malignant cancer.Cancers or tumors include but are not limited to biliary tract cancer;brain cancer; breast cancer; cervical cancer; choriocarcinoma; coloncancer; endometrial cancer; esophageal cancer; gastric cancer;intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g.small cell and non-small cell); melanoma; neuroblastomas; oral cancer;ovarian cancer; pancreas cancer; prostate cancer; rectal cancer;sarcomas; skin cancer; testicular cancer; thyroid cancer; and renalcancer, as well as other carcinomas and sarcomas.

[0051] A “subject” shall mean a human or vertebrate animal including butnot limited to a dog, cat, horse, cow, pig, sheep, goat, chicken,primate, (e.g., monkey), fish (aquaculture species e.g. salmon, troutand other salmonids), rat, and mouse.

[0052] The subject is administered a combination of adjuvants, whereinthe combination of adjuvants includes at least one oligonucleotidecontaining at least one unmethylated CpG dinucleotide and at least onenon-nucleic acid adjuvant. An oligonucleotide containing at least oneunmethylated CpG dinucleotide is a nucleic acid molecule which containsan unmethylated cytosine-guanine dinucleotide sequence (i.e. “CpG DNA”or DNA containing a 5′ cytosine followed by 3′ guanosine and linked by aphosphate bond) and activates the immune system. The CpGoligonucleotides can be double-stranded or single-stranded. Generally,double-stranded molecules are more stable in vivo, while single-strandedmolecules have increased immune activity. The CpG oligonucleotides orcombination of adjuvants can be used with or without antigen.

[0053] The terms “nucleic acid” and “oligonucleotide” are usedinterchangeably to mean multiple nucleotides (i.e. molecules comprisinga sugar (e.g. ribose or deoxyribose) linked to a phosphate group and toan exchangeable organic base, which is either a substituted pyrimidine(e.g. cytosine (C), thymine (T) or uracil (U)) or a substituted purine(e.g. adenine (A) or guanine (G)). As used herein, the terms refer tooligoribonucleotides as well as oligodeoxyribonucleotides. The termsshall also include polynucleosides (i.e. a polynucleotide minus thephosphate) and any other organic base containing polymer. Nucleic acidmolecules can be obtained from existing nucleic acid sources (e.g.genomic or cDNA), but are preferably synthetic (e.g. produced byoligonucleotide synthesis). The entire CpG oligonucleotide can beunmethylated or portions may be unmethylated but at least the C of the5′ CG 3′ must be unmethylated.

[0054] In one preferred embodiment the invention provides a CpGoligonucleotide represented by at least the formula:

5′N₁X₁CGX₂N₂3′

[0055] wherein at least one nucleotide separates consecutive CpGs; X₁ isadenine, guanine, or thymine; X₂ is cytosine, adenine, or thymine; N isany nucleotide and N₁ and N₂ are nucleic acid sequences composed of fromabout 0-25 N's each.

[0056] In another embodiment the invention provides an isolated CpGoligonucleotide represented by at least the formula:

5′N₁X₁X₂CGX₃X₄N₂3′

[0057] wherein at least one nucleotide separates consecutive CpGs; X₁X₂are nucleotides selected from the group consisting of: GpT, GpG, GpA,ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and X₃X₄ arenucleotides selected from the group consisting of: TpT, CpT, ApT, TpG,ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA; N is any nucleotide and N₁and N₂ are nucleic acid sequences composed of from about 0-25 N's each.Preferably X₁X₂ are GpA or GpT and X₃X₄ are TpT. In other preferredembodiments X₁ or X₂ or both are purines and X₃ or X₄ or both arepyrimidines or X₁X₂ are GpA and X₃ or X₄ or both are pyrimidines. In apreferred embodiment N₁ and N₂ of the nucleic acid do not contain a CCGGor CGCG quadmer or more than one CCG or CGG trimer. The effect of a aCCGG or CGCG quadmer or more than one CCG or CGG trimer depends in parton the status of the oligonucleotide backbone. For instance, if theoligonucleotide has a phosphodiester backbone or a chimeric backbone theinclusion of these sequences in the oligonucleotide will only haveminimal if any affect on the biological activity of the oligonucleotide.If the backbone is completely phosphorothioate or significantlyphosphorothioate then the inclusion of these sequences may have moreinfluence on the biological activity or the kinetics of the biologicalactivity. In another preferred embodiment the CpG oligonucleotide hasthe sequence 5′TCN₁TX₁X₂CGX₃X₄3′.

[0058] Preferably the CpG oligonucleotides of the invention include X₁X₂selected from the group consisting of GpT, GpG, GpA and ApA and X₃X₄ isselected from the group consisting of TpT, CpT and GpT. For facilitatinguptake into cells, CpG containing oligonucleotides are preferably in therange of 8 to 30 bases in length. However, nucleic acids of any sizegreater than 8 nucleotides (even many kb long) are capable of inducingan immune response according to the invention if sufficientimmunostimulatory motifs are present, since larger nucleic acids aredegraded into oligonucleotides inside of cells. Preferred syntheticoligonucleotides do not include a CCGG or CGCG quadmer or more than oneCCG or CGG trimer at or near the 5′ and/or 3′ terminals. Stabilizedoligonucleotides, where the oligonucleotide incorporates a phosphatebackbone modification, as discussed in more detail below are alsopreferred. The modification may be, for example, a phosphorothioate orphosphorodithioate modification. Preferably, the phosphate backbonemodification occurs at the 5′ end of the nucleic acid for example, atthe first two nucleotides of the 5′ end of the oligonucleotide. Further,the phosphate backbone modification may occur at the 3′ end of thenucleic acid for example, at the last five nucleotides of the 3′ end ofthe nucleic acid. Alternatively the oligonucleotide may be completely orpartially modified.

[0059] Preferably the CpG oligonucleotide is in the range of between 8and 100 and more preferably between 8 and 30 nucleotides in size.Alternatively, CpG oligonucleotides can be produced on a large scale inplasmids. These may be administered in plasmid form or alternativelythey can be degraded into oligonucleotides.

[0060] The CpG oligonucleotide and immunopotentiating cytokine may bedirectly administered to the subject or may be administered inconjunction with a nucleic acid delivery complex. A “nucleicacid/cytokine delivery complex” shall mean a nucleic acid moleculeand/or cytokine associated with (e.g. ionically or covalently bound to;or encapsulated within) a targeting means (e.g. a molecule that resultsin higher affinity binding to target cell (e.g. dendritic cell surfacesand/or increased cellular uptake by target cells). Examples of nucleicacid/cytokine delivery complexes include nucleic acids/cytokinesassociated with: a sterol (e.g. cholesterol), a lipid (e.g. a cationiclipid, virosome or liposome), or a target cell specific binding agent(e.g. a ligand recognized by target cell specific receptor). Preferredcomplexes should be sufficiently stable in vivo to prevent significantuncoupling prior to internalization by the target cell. However, thecomplex should be cleavable under appropriate conditions within the cellso that the nucleic acid/cytokine is released in a functional form.

[0061] “Palindromic sequence” shall mean an inverted repeat (i.e. asequence such as ABCDEE′D′C′B′A′ in which A and A′ are bases capable offorming the usual Watson-Crick base pairs. In vivo, such sequences mayform double-stranded structures. In one embodiment the CpGoligonucleotide contains a palindromic sequence. A palindromic sequenceused in this context refers to a palindrome in which the CpG is part ofthe palindrome, and preferably is the center of the palindrome. Inanother embodiment the CpG oligonucleotide is free of a palindrome. ACpG oligonucleotide that is free of a palindrome is one in which the CpGdinucleotide is not part of a palindrome. Such an oligonucleotide mayinclude a palindrome in which the CpG is not part of the palindrome.

[0062] A “stabilized nucleic acid molecule” shall mean a nucleic acidmolecule that is relatively resistant to in vivo degradation (e.g. viaan exo- or endo-nuclease). Stabilization can be a function of length orsecondary structure. Unmethylated CpG oligonucleotides that are tens tohundreds of kbs long are relatively resistant to in vivo degradation,particularly when in a double-stranded closed-circular form (i.e., aplamid). For shorter CpG oligonucleotides, secondary structure canstabilize and increase their effect. For example, if the 3′ end of anoligonucleotide has self-complementarity to an upstream region, so thatit can fold back and form a sort of stem loop structure, then theoligonucleotide becomes stabilized and therefore exhibits more activity.

[0063] Preferred stabilized oligonucleotides of the instant inventionhave a modified backbone. It has been demonstrated that modification ofthe oligonucleotide backbone provides enhanced activity of the CpGoligonucleotides when administered in vivo. CpG constructs, including atleast two phosphorothioate linkages at the 5′ end of the oligonucleotidein multiple phosphorothioate linkages at the 3′ end, preferably 5,provides maximal activity and protected the oligonucleotide fromdegradation by intracellular exo- and endo-nucleases. Other modifiedoligonucleotides include phosphodiester modified oligonucleotide,combinations of phosphodiester and phosphorothioate oligonucleotide,methylphosphonate, methylphosphorothioate, phosphorodithioate, andcombinations thereof. Each of these combinations and their particulareffects on immune cells is discussed in more detail in copending PCTPublished Patent Applications claiming priority to U.S. Ser. Nos.08/738,652 and 08/960,774, filed on Oct. 30, 1996 and Oct. 30, 1997respectively, the entire contents of which is hereby incorporated byreference. It is believed that these modified oligonucleotides may showmore stimulatory activity due to enhanced nuclease resistance, increasedcellular uptake, increased protein binding, and/or altered intracellularlocalization.

[0064] Both phosphorothioate and phosphodiester oligonucleotidescontaining CpG motifs are active in immune cells. However, based on theconcentration needed to induce CpG specific effects, the nucleaseresistant phosphorothioate backbone CpG oligonucleotides are more potent(2 μg/ml for the phosphorothioate vs. a total of 90 μg/ml forphosphodiester).

[0065] Other stabilized oligonucleotides include: nonionic DNA analogs,such as alkyl- and aryl-phosphates (in which the charged phosphonateoxygen is replaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Oligonucleotides which contain diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation.

[0066] The nucleic acid sequences of the invention which are useful asadjuvants are those broadly described above and disclosed in PCTPublished Patent Applications claiming priority to U.S. Ser. Nos.08/738,652 and 08/960,774, filed on Oct. 30, 1996 and Oct. 30, 1997respectively. Exemplary sequences include but are not limited to thoseimmunostimulatory sequences shown in Table 1.

[0067] The stimulation index of a particular immunostimulatory CpG DNAcan be tested in various immune cell assays. Preferably, the stimulationindex of the CpG oligonucleotide with regard to B cell proliferation isat least about 5, preferably at least about 10, more preferably at leastabout 15 and most preferably at least about 20 as determined byincorporation of ³H uridine in a murine B cell culture, which has beencontacted with 20 μM of oligonucleotide for 20 h at 37° C. and has beenpulsed with 1 μCi of ³H uridine; and harvested and counted 4 h later asdescribed in detail in copending PCT Published Patent Applicationsclaiming priority to U.S. Ser. Nos. 08/738,652 and 08/960,774, filed onOct. 30, 1996 and Oct. 30, 1997 respectively. For use in vivo, forexample, it is important that the CpG oligonucleotide and adjuvant becapable of effectively inducing activation of Ig expressing B cells.Oligonucleotides which can accomplish this include, for example, but arenot limited to those oligonucleotides described in PCT Published PatentApplications claiming priority to U.S. Ser. Nos. 08/738,652 and08/960,774, filed on Oct. 30, 1996 and Oct. 30, 1997 respectively.

[0068] The oligonucleotide containing at least one unmethylated CpG isused in combination with a non-nucleic acid adjuvant and an antigen toactivate the immune response. A “non-nucleic acid adjuvant” is anymolecule or compound except for the CpG oligonucleotides describedherein which can stimulate the humoral and/or cellular immune response.Non-nucleic acid adjuvants include, for instance, adjuvants that createa depo effect, immune stimulating adjuvants, and adjuvants that create adepo effect and stimulate the immune system. In infants, theoligonucleotide containing at least one unmethylated CpG is used aloneor in combination with a non-nucleic acid adjuvant and an antigen toactivate a cellular immune response.

[0069] An “adjuvant that creates a depo effect” as used herein is anadjuvant that causes the antigen to be slowly released in the body, thusprolonging the exposure of immune cells to the antigen. This class ofadjuvants includes but is not limited to alum (e.g., aluminum hydroxide,aluminum phosphate); or emulsion-based formulations including mineraloil, non-mineral oil, water-in-oil or oil-in-water-in oil emulsion,oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants(e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (asqualene-in-water emulsion stabilized with Span 85 and Tween 80; ChironCorporation, Emeryville, Calif.; and PROVAX (an oil-in-water emulsioncontaining a stabilizing detergent and a micelle-forming agent; IDEC,Pharmaceuticals Corporation, San Diego, Calif.).

[0070] An “immune stimulating adjuvant” is an adjuvant that causesactivation of a cell of the immune system. It may, for instance, causean immune cell to produce and secrete cytokines. This class of adjuvantsincludes but is not limited to saponins purified from the bark of the Q.saponaria tree, such as QS21 (a glycolipid that elutes in the 21^(st)peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc.,Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer;Virus Research Institute, USA); derivatives of lipopolysaccharides suchas monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc.,Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyldipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related tolipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongationfactor (a purified Leishmania protein; Corixa Corporation, Seattle,Wash.).

[0071] “Adjuvants that create a depo effect and stimulate the immunesystem” are those compounds which have both of the above-identifiedfunctions. This class of adjuvants includes but is not limited to ISCOMS(Immunostimulating complexes which contain mixed saponins, lipids andform virus-sized particles with pores that can hold antigen; CSL,Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2which is an oil-in-water emulsion containing MPL and QS21: SmithKlineBeecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKlineBeecham adjuvant system #4 which contains alum and MPL; SBB, Belgium);non-ionic block copolymers that form micelles such as CRL 1005 (thesecontain a linear chain of hydrophobic polyoxpropylene flanked by chainsof polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex AdjuvantFormulation (SAF, an oil-in-water emulsion containing Tween 80 and anonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.).

[0072] When the CpG oligonucleotide containing at least one unmethylatedCpG is administered in conjunction with another adjuvant, the CpGoligonucleotide can be administered before, after, and/or simultaneouslywith the other adjuvant. For instance, the combination of adjuvants maybe administered with a priming dose of antigen. Either or both of theadjuvants may then be administered with the boost dose. Alternatively,the combination of adjuvants may be administered with a boost dose ofantigen. Either or both of the adjuvants may then be administered withthe prime dose. A “prime dose” is the first dose of antigen administeredto the subject. In the case of a subject that has an infection the primedose may be the initial exposure of the subject to the infectiousmicrobe and thus the combination of adjuvants is administered to thesubject with the boost dose. A “boost dose” is a second or third, etc.,dose of antigen administered to a subject that has already been exposedto the antigen. In some cases the prime dose administered with thecombination of adjuvants is so effective that a boost dose is notrequired to protect a subject at risk of infection from being infected.In cases where the combination of adjuvants is given without antigen,with repeated administrations, CpG oligonucleotides or one of thecomponents in the combination may be given alone for one or more of theadministrations.

[0073] The CpG oligonucleotide containing at least one unmethylated CpGcan have an additional efficacy (e.g., antisense) in addition to itsability to enhance antigen-specific immune responses.

[0074] An “antigen” as used herein is a molecule capable of provoking animmune response. Antigens include but are not limited to peptides,polypeptides, cells, cell extracts, polysaccharides, polysaccharideconjugates, lipids, glycoliopids and carbohydrates. Antigens may begiven in a crude, purified or recombinant form and polypeptide/peptideantigens, including peptide mimics of polysaccharides, may also beencoded within nucleic. The term antigen broadly includes any type ofmolecule which is recognized by a host immune system as being foreign.Antigens include but are not limited to cancer antigens, microbialantigens, and allergens.

[0075] A “cancer antigen” as used herein is a compound, such as apeptide, associated with a tumor or cancer cell surface and which iscapable of provoking an immune response when expressed on the surface ofan antigen presenting cell in the context of an MHC molecule. Cancerantigens can be prepared from cancer cells either by preparing crudeextracts of cancer cells, for example, as described in Cohen, et al.,1994, Cancer Research, 54:1055, by partially purifying the antigens, byrecombinant technology, or by de novo synthesis of known antigens.Cancer antigens include antigens that are recombinately an immunogenicportion of or a whole tumor or cancer. Such antigens can be isolated orprepared recombinatly or by any other means known in the art.

[0076] A “microbial antigen” as used herein is an antigen of amicroorganism and includes but is not limited to infectious virus,infectious bacteria, infectious parasites and infectious fungi. Suchantigens include the intact microorganism as well as natural isolatesand fragments or derivatives thereof and also synthetic compounds whichare identical to or similar to natural microorganism antigens and inducean immune response specific for that microorganism. A compound issimilar to a natural microorganism antigen if it induces an immuneresponse (humoral and/or cellular) to a natural microorganism antigen.Most such antigens are used routinely in the art and are well known tothose of ordinary skill in the art. Another example is a peptide mimicof a polysaccharide antigen.

[0077] Examples of infectious virus that have been found in humansinclude but are not limited to: Retroviridae (e.g. humanimmunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III,LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.strains that cause gastroenteritis); Togaviridae (e.g. equineencephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses,encephalitis viruses, yellow fever viruses); Coronoviridae (e.g.coronaviruses); Rhabdoviradae (e.g. vesicular stomatitis viruses, rabiesviruses); Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g.vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebolaviruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g. African swine fever virus); and unclassified viruses(e.g. the etiological agents of Spongiform encephalopathies, the agentof delta hepatitis (thought to be a defective satellite of hepatitis Bvirus), the agents of non-A, non-B hepatitis (class 1=internallytransmitted; class 2=parenterally transmitted (i.e. Hepatitis C);Norwalk and related viruses, and astroviruses).

[0078] Both gram negative and gram positive bacteria serve as antigensin vertebrate animals. Such gram positive bacteria include, but are notlimited to Pasteurella species, Staphylococci species, and Streptococcusspecies. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species, and Salmonella species. Specificexamples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus monilijormis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

[0079] Examples of infectious fungi include: Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,Chlamydia trachomatis, Candida albicans. Examples of infectiousparasites include Plasmodium such as Plasmodium falciparum, Plasmodiummalariae, Plasmodium ovale, and Plasmodium vivax. Other infectiousorganisms (i.e. protists) include Toxoplasma gondii.

[0080] Other medically relevant microorganisms have been descriedextensively in the literature, e.g., see C. G. A Thomas, MedicalMicrobiology, Bailliere Tindall, Great Britain 1983, the entire contentsof which is hereby incorporated by reference.

[0081] Although many of the microbial antigens described above relate tohuman disorders, the invention is also useful for treating othernonhuman vertebrates. Nonhuman vertebrates are also capable ofdeveloping infections which can be prevented or treated with thesynergistic combination of adjuvants disclosed herein. For instance, inaddition to the treatment of infectious human diseases, the methods ofthe invention are useful for treating infections of animals.

[0082] As used herein, the term “treat”, “treated”, or “treating” whenused with respect to an infectious disease refers to a prophylactictreatment which increases the resistance of a subject (a subject at riskof infection) to infection with a pathogen or, in other words, decreasesthe likelihood that the subject will become infected with the pathogen,as well as a treatment after the subject (a subject who has beeninfected) has become infected in order to fight the infection, e.g.,reduce or eliminate the infection or prevent it from becoming worse.Many vaccines for the treatment of non-human vertebrates are disclosedin Bennett, K. Compendium of Veterinary Products, 3rd ed. North AmericanCompendiums, Inc., 1995.

[0083] As discussed above, antigens include infectious microbes such asvirus, bacteria, parasites and fungi and fragments thereof, derived fromnatural sources or synthetically. Infectious virus of both human andnon-human vertebrates, include retroviruses, RNA viruses and DNAviruses. This group of retroviruses includes both simple retrovirusesand complex retroviruses. The simple retroviruses include the subgroupsof B-type retroviruses, C-type retroviruses and D-type retroviruses. Anexample of a B-type retrovirus is mouse mammary tumor virus (MMTV). TheC-type retroviruses include subgroups C-type group A (including Roussarcoma virus (RSV), avian leukemia virus (ALV), and avianmyeloblastosis virus (AMV)) and C-type group B (including murineleukemia virus (MLV), feline leukemia virus (FeLV), murine sarcoma virus(MSV), gibbon ape leukemia virus (GALV), spleen necrosis virus (SNV),reticuloendotheliosis virus (RV) and simian sarcoma virus (SSV)). TheD-type retroviruses include Mason-Pfizer monkey virus (MPMV) and simianretrovirus type 1 (SRV-1). The complex retroviruses include thesubgroups of lentiviruses, T-cell leukemia viruses and the foamyviruses. Lentiviruses include HIV-1, but also include HIV-2, SIV, Visnavirus, feline immunodeficiency virus (FIV), and equine infectious anemiavirus (EIAV). The T-cell leukemia viruses include HTLV-1, HTLV-II,simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV).The foamy viruses include human foamy virus (HFV), simian foamy virus(SFV) and bovine foamy virus (BFV).

[0084] Examples of other RNA viruses that are antigens in vertebrateanimals include, but are not limited to, the following: members of thefamily Reoviridae, including the genus Orthoreovirus (multiple serotypesof both mammalian and avian retroviruses), the genus Orbivirus(Bluetongue virus, Eugenangee virus, Kemerovo virus, African horsesickness virus, and Colorado Tick Fever virus), the genus Rotavirus(human rotavirus, Nebraska calf diarrhea virus, murine rotavirus, simianrotavirus, bovine or ovine rotavirus, avian rotavirus); the familyPicornaviridae, including the genus Enterovirus (poliovirus, Coxsackievirus A and B, enteric cytopathic human orphan (ECHO) viruses, hepatitisA virus, Simian enteroviruses, Murine encephalomyelitis (ME) viruses,Poliovirus muris, Bovine enteroviruses, Porcine enteroviruses, the genusCardiovirus (Encephalomyocarditis virus (EMC), Mengovirus), the genusRhinovirus (Human rhinoviruses including at least 113 subtypes; otherrhinoviruses), the genus Apthovirus (Foot and Mouth disease (FMDV); thefamily Calciviridae, including Vesicular exanthema of swine virus, SanMiguel sea lion virus, Feline picornavirus and Norwalk virus; the familyTogaviridae, including the genus Alphavirus (Eastern equine encephalitisvirus, Semliki forest virus, Sindbis virus, Chikungunya virus,O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitisvirus, Western equine encephalitis virus), the genus Flavirius (Mosquitoborne yellow fever virus, Dengue virus, Japanese encephalitis virus, St.Louis encephalitis virus, Murray Valley encephalitis virus, West Nilevirus, Kunjin virus, Central European tick borne virus, Far Eastern tickborne virus, Kyasanur forest virus, Louping III virus, Powassan virus,Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), thegenus Pestivirus (Mucosal disease virus, Hog cholera virus, Borderdisease virus); the family Bunyaviridae, including the genus Bunyvirus(Bunyamwera and related viruses, California encephalitis group viruses),the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fevervirus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus,Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi andrelated viruses); the family Orthomyxoviridae, including the genusInfluenza virus (Influenza virus type A, many human subtypes); Swineinfluenza virus, and Avian and Equine Influenza viruses; influenza typeB (many human subtypes), and influenza type C (possible separate genus);the family paramyxoviridae, including the genus Paramyxovirus(Parainfluenza virus type 1, Sendai virus, Hemadsorption virus,Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumpsvirus), the genus Morbillivirus (Measles virus, subacute sclerosingpanencephalitis virus, distemper virus, Rinderpest virus), the genusPneumovirus (respiratory syncytial virus (RSV), Bovine respiratorysyncytial virus and Pneumonia virus of mice); forest virus, Sindbisvirus, Chikungunya virus, O'Nyong-Nyong virus, Ross river virus,Venezuelan equine encephalitis virus, Western equine encephalitisvirus), the genus Flavirius (Mosquito borne yellow fever virus, Denguevirus, Japanese encephalitis virus, St. Louis encephalitis virus, MurrayValley encephalitis virus, West Nile virus, Kunjin virus, CentralEuropean tick borne virus, Far Eastern tick borne virus, Kyasanur forestvirus, Louping III virus, Powassan virus, Omsk hemorrhagic fever virus),the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosaldisease virus, Hog cholera virus, Border disease virus); the familyBunyaviridae, including the genus Bunyvirus (Bunyamwera and relatedviruses, California encephalitis group viruses), the genus Phlebovirus(Sandfly fever Sicilian virus, Rift Valley fever virus), the genusNairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep diseasevirus), and the genus Uukuvirus (Uukuniemi and related viruses); thefamily Orthomyxoviridae, including the genus Influenza virus (Influenzavirus type A, many human subtypes); Swine influenza virus, and Avian andEquine Influenza viruses; influenza type B (many human subtypes), andinfluenza type C (possible separate genus); the family paramyxoviridae,including the genus Paramyxovirus (Parainfluenza virus type 1, Sendaivirus, Hemadsorption virus, Parainfluenza viruses types 2 to 5,Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measlesvirus, subacute sclerosing panencephalitis virus, distemper virus,Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus(RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice);the family Rhabdoviridae, including the genus Vesiculovirus (VSV),Chandipura virus, Flanders-Hart Park virus), the genus Lyssavirus(Rabies virus), fish Rhabdoviruses, and two probable Rhabdoviruses(Marburg virus and Ebola virus); the family Arenaviridae, includingLymphocytic choriomeningitis virus (LCM), Tacaribe virus complex, andLassa virus; the family Coronoaviridae, including Infectious BronchitisVirus (IBV), Mouse Hepatitis virus, Human enteric corona virus, andFeline infectious peritonitis (Feline coronavirus).

[0085] Illustrative DNA viruses that are antigens in vertebrate animalsinclude, but are not limited to: the family Poxviridae, including thegenus Orthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia,Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus(Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avianpoxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genusSuipoxvirus (Swinepox), the genus Parapoxvirus (contagious postulardermatitis virus, pseudocowpox, bovine papular stomatitis virus); thefamily Iridoviridae (African swine fever virus, Frog viruses 2 and 3,Lymphocystis virus of fish); the family Herpesviridae, including thealpha-Herpesviruses (Herpes Simplex Types 1 and 2, Varicella-Zoster,Equine abortion virus, Equine herpes virus 2 and 3, pseudorabies virus,infectious bovine keratoconjunctivitis virus, infectious bovinerhinotracheitis virus, feline rhinotracheitis virus, infectiouslaryngotracheitis virus) the Beta-herpesviruses (Human cytomegalovirusand cytomegaloviruses of swine, monkeys and rodents); thegamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease virus,Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus, guinea pigherpes virus, Lucke tumor virus); the family Adenoviridae, including thegenus Mastadenovirus (Human subgroups A,B,C,D,E and ungrouped; simianadenoviruses (at least 23 serotypes), infectious canine hepatitis, andadenoviruses of cattle, pigs, sheep, frogs and many other species, thegenus Aviadenovirus (Avian adenoviruses); and non-cultivatableadenoviruses; the family Papoviridae, including the genus Papillomavirus(Human papilloma viruses, bovine papilloma viruses, Shope rabbitpapilloma virus, and various pathogenic papilloma viruses of otherspecies), the genus Polyomavirus (polyomavirus, Simian vacuolating agent(SV-40), Rabbit vacuolating agent (RKV), K virus, BK virus, JC virus,and other primate polyoma viruses such as Lymphotrophic papillomavirus); the family Parvoviridae including the genus Adeno-associatedviruses, the genus Parvovirus (Feline panleukopenia virus, bovineparvovirus, canine parvovirus, Aleutian mink disease virus, etc).Finally, DNA viruses may include viruses which do not fit into the abovefamilies such as Kuru and Creutzfeldt-Jacob disease viruses and chronicinfectious neuropathic agents (CHINA virus).

[0086] Each of the foregoing lists is illustrative, and is not intendedto be limiting.

[0087] In addition to the use of the combination of CpG oligonucleotidesand non-nucleic acid adjuvants to induce an antigen specific immuneresponse in humans, the methods of the preferred embodiments areparticularly well suited for treatment of birds such as hens, chickens,turkeys, ducks, geese, quail, and pheasant. Birds are prime targets formany types of infections.

[0088] Hatching birds are exposed to pathogenic microorganisms shortlyafter birth. Although these birds are initially protected againstpathogens by maternal derived antibodies, this protection is onlytemporary, and the bird's own immature immune system must begin toprotect the bird against the pathogens. It is often desirable to preventinfection in young birds when they are most susceptible. It is alsodesirable to prevent against infection in older birds, especially whenthe birds are housed in closed quarters, leading to the rapid spread ofdisease. Thus, it is desirable to administer the CpG oligonucleotide andthe non-nucleic acid adjuvant of the invention to birds to enhance anantigen-specific immune response when antigen is present. The CpGoligonucleotide and the non-nucleic acid adjuvant of the invention couldalso be administered to birds without antigen to protect againstinfection of a wide variety of pathogens.

[0089] An example of a common infection in chickens is chickeninfectious anemia virus (CIAV). CIAV was first isolated in Japan in 1979during an investigation of a Marek's disease vaccination break (Yuasa etal., 1979, Avian Dis. 23:366-385). Since that time, CIAV has beendetected in commercial poultry in all major poultry producing countries(van Bulow et al., 1991, pp.690-699) in Diseases of Poultry, 9thedition, Iowa State University Press).

[0090] CIAV infection results in a clinical disease, characterized byanemia, hemorrhage and immunosuppression, in young susceptible chickens.Atrophy of the thymus and of the bone marrow and consistent lesions ofCIAV-infected chickens are also characteristic of CIAV infection.Lymphocyte depletion in the thymus, and occasionally in the bursa ofFabricius, results in immunosuppression and increased susceptibility tosecondary viral, bacterial, or fungal infections which then complicatethe course of the disease. The immunosuppression may cause aggravateddisease after infection with one or more of Marek's disease virus (MDV),infectious bursal disease virus, reticuloendotheliosis virus,adenovirus, or reovirus. It has been reported that pathogenesis of MDVis enhanced by CIAV (DeBoer et al., 1989, p. 28 In Proceedings of the38th Western Poultry Diseases Conference, Tempe, Ariz.). Further, it hasbeen reported that CIAV aggravates the signs of infectious bursaldisease (Rosenberger et al., 1989, Avian Dis. 33:707-713). Chickensdevelop an age resistance to experimentally induced disease due to CAA.This is essentially complete by the age of 2 weeks, but older birds arestill susceptible to infection (Yuasa, N. et al., 1979 supra; Yuasa, N.et al., Arian Diseases 24, 202-209, 1980). However, if chickens aredually infected with CAA and an immunosuppressive agent (IBDV, MDV etc.)age resistance against the disease is delayed (Yuasa, N. et al., 1979and 1980 supra; Bulow von V. et al., J. Veterinary Medicine 33, 93-116,1986). Characteristics of CIAV that may potentiate disease transmissioninclude high resistance to environmental inactivation and some commondisinfectants. The economic impact of CIAV infection on the poultryindustry is clear from the fact that 10% to 30% of infected birds indisease outbreaks die.

[0091] Vaccination of birds, like other vertebrate animals can beperformed at any age. Normally, vaccinations are performed at up to 12weeks of age for a live microorganism and between 14-18 weeks for aninactivated microorganism or other type of vaccine. For in ovovaccination, vaccination can be performed in the last quarter of embryodevelopment. The vaccine may be administered subcutaneously, by spray,orally, intraocularly, intratracheally, nasally, in ovo or by othermethods described herein. Thus, the CpG oligonucleotide and non-nucleicacid adjuvant of the invention can be administered to birds and othernon-human vertebrates using routine vaccination schedules and theantigen is administered after an appropriate time period as describedherein.

[0092] Cattle and livestock are also susceptible to infection. Diseasewhich affect these animals can produce severe economic losses,especially amongst cattle. The methods of the invention can be used toprotect against infection in livestock, such as cows, horses, pigs,sheep, and goats. The CpG oligonucleotide and the non-nucleic acidadjuvant of the invention could also be administered with antigen forantigen-specific protection of long duration or without antigen forshort term protection against a wide variety of diseases, includingshipping fever.

[0093] Cows can be infected by bovine viruses. Bovine viral diarrheavirus (BVDV) is a small enveloped positive-stranded RNA virus and isclassified, along with hog cholera virus (HOCV) and sheep border diseasevirus (BDV), in the pestivirus genus. Although, Pestiviruses werepreviously classified in the Togaviridae family, some studies havesuggested their reclassification within the Flaviviridae family alongwith the flavivirus and hepatitis C virus (HCV) groups (Francki, et al.,1991).

[0094] BVDV, which is an important pathogen of cattle can bedistinguished, based on cell culture analysis, into cytopathogenic (CP)and noncytopathogenic (NCP) biotypes. The NCP biotype is more widespreadalthough both biotypes can be found in cattle. If a pregnant cow becomesinfected with an NCP strain, the cow can give birth to a persistentlyinfected and specifically immunotolerant calf that will spread virusduring its lifetime. The persistently infected cattle can succumb tomucosal disease and both biotypes can then be isolated from the animal.Clinical manifestations can include abortion, teratogenesis, andrespiratory problems, mucosal disease and mild diarrhea. In addition,severe thrombocytopenia, associated with herd epidemics, that may resultin the death of the animal has been described and strains associatedwith this disease seem more virulent than the classical BVDVs.

[0095] Equine herpesviruses (EHV) comprise a group of antigenicallydistinct biological agents which cause a variety of infections in horsesranging from subclinical to fatal disease. These include Equineherpesvirus-1 (EHV-1), a ubiquitous pathogen in horses. EHV-1 isassociated with epidemics of abortion, respiratory tract disease, andcentral nervous system disorders. Primary infection of upper respiratorytract of young horses results in a febrile illness which lasts for 8 to10 days. Immunologically experienced mares may be reinfected via therespiratory tract without disease becoming apparent, so that abortionusually occurs without warning. The neurological syndrome is associatedwith respiratory disease or abortion and can affect animals of eithersex at any age, leading to incoordination, weakness and posteriorparalysis (Telford, E. A. R. et al., Virology 189, 304-316, 1992). OtherEHV's include EHV-2, or equine cytomegalovirus, EHV-3, equine coitalexanthema virus, and EHV-4, previously classified as EHV-1 subtype 2.

[0096] Sheep and goats can be infected by a variety of dangerousmicroorganisms including visna-maedi.

[0097] Primates such as monkeys, apes and macaques can be infected bysimian immunodeficiency virus. Inactivated cell-virus and cell-freewhole simian immunodeficiency vaccines have been reported to affordprotection in macaques (Stott et al. (1990) Lancet 36:1538-1541;Desrosiers et al. PNAS USA (1989) 86:6353-6357; Murphey-Corb et al.(1989) Science 246:1293-1297; and Carlson et al. (1990) AIDS Res. HumanRetroviruses 6:1239-1246). A recombinant HIV gp120 vaccine has beenreported to afford protection in chimpanzees (Berman et al. (1990)Nature 345:622-625).

[0098] Cats, both domestic and wild, are susceptible to infection with avariety of microorganisms. For instance, feline infectious peritonitisis a disease which occurs in both domestic and wild cats, such as lions,leopards, cheetahs, and jaguars. When it is desirable to preventinfection with this and other types of pathogenic organisms in cats, themethods of the invention can be used to vaccinate cats to prevent themagainst infection.

[0099] Domestic cats may become infected with several retroviruses,including but not limited to feline leukemia virus (FeLV), felinesarcoma virus (FeSV), endogenous type C oncornavirus (RD-114), andfeline syncytia-forming virus (FeSFV). Of these, FeLV is the mostsignificant pathogen, causing diverse symptoms, includinglymphoreticular and myeloid neoplasms, anemias, immune mediateddisorders, and an immunodeficiency syndrome which is similar to humanacquired immune deficiency syndrome (AIDS). Recently, a particularreplication-defective FeLV mutant, designated FeLV-AIDS, has been moreparticularly associated with immunosuppressive properties.

[0100] The discovery of feline T-lymphotropic lentivirus (also referredto as feline immunodeficiency) was first reported in Pedersen et al.(1987) Science 235:790-793. Characteristics of FIV have been reported inYamamoto et al. (1988) Leukemia, December Supplement 2:204S-215S;Yamamoto et al. (1988) Am. J. Vet. Res. 49:1246-1258; and Ackley et al.(1990) J. Virol. 64:5652-5655. Cloning and sequence analysis of FIV havebeen reported in Olmsted et al. (1989) Proc. Natl. Acad. Sci. USA86:2448-2452 and 86:4355-4360.

[0101] Feline infectious peritonitis (FIP) is a sporadic diseaseoccurring unpredictably in domestic and wild Felidae. While FIP isprimarily a disease of domestic cats, it has been diagnosed in lions,mountain lions, leopards, cheetahs, and the jaguar. Smaller wild catsthat have been afflicted with FIP include the lynx and caracal, sandcat, and pallas cat. In domestic cats, the disease occurs predominantlyin young animals, although cats of all ages are susceptible. A peakincidence occurs between 6 and 12 months of age. A decline in incidenceis noted from 5 to 13 years of age, followed by an increased incidencein cats 14 to 15 years old.

[0102] Viral, bacterial and parasitic diseases in fin-fish, shellfish orother aquatic life forms pose a serious problem for the aquacultureindustry. Owing to the high density of animals in the hatchery tanks orenclosed marine farming areas, infectious diseases may eradicate a largeproportion of the stock in, for example, a fin-fish, shellfish, or otheraquatic life forms facility. Prevention of disease is a more desiredremedy to these threats to fish than intervention once the disease is inprogress. Vaccination of fish is the only preventative method which mayoffer long-term protection through immunity. Fish are currentlyprotected against a variety of bacterial infections with whole killedvaccines with oli adjuvants, but there is only one licensed vaccine forfish against a viral disease. Nucleic acid based vaccinations aredescribed in U.S. Pat. No. 5,780,448 issued to Davis and these have beenshown to be protective against at least two different viral diseases.

[0103] The fish immune system has many features similar to the mammalianimmune system, such as the presence of B cells, T cells, lymphokines,complement, and immunoglobulins. Fish have lymphocyte subclasses withroles that appear similar in many respects to those of the B and T cellsof mammals. Vaccines can be administered orally or by immersion orinjection.

[0104] Aquaculture species include but are not limited to fin-fish,shellfish, and other aquatic animals. Fin-fish include all vertebratefish, which may be bony or cartilaginous fish, such as, for example,salmonids, carp, catfish, yellowtail, seabream, and seabass. Salmonidsare a family of fin-fish which include trout (including rainbow trout),salmon, and Arctic char. Examples of shellfish include, but are notlimited to, clams, lobster, shrimp, crab, and oysters. Other culturedaquatic animals include, but are not limited to eels, squid, and octopi.

[0105] Polypeptides of viral aquaculture pathogens include but are notlimited to glycoprotein (G) or nucleoprotein (N) of viral hemorrhagicsepticemia virus (VHSV); G or N proteins of infectious hematopoieticnecrosis virus (IHNV); VP1, VP2, VP3 or N structural proteins ofinfectious pancreatic necrosis virus (IPNV); G protein of spring viremiaof carp (SVC); and a membrane-associated protein, tegumin or capsidprotein or glycoprotein of channel catfish virus (CCV).

[0106] Polypeptides of bacterial pathogens include but are not limitedto an iron-regulated outer membrane protein, (IROMP), an outer membraneprotein (OMP), and an A-protein of Aeromonis salmonicida which causesfurunculosis, p57 protein of Renibacterium salmoninarum which causesbacterial kidney disease (BKD), major surface associated antigen (msa),a surface expressed cytotoxin (mpr), a surface expressed hemolysin(ish), and a flagellar antigen of Yersiniosis; an extracellular protein(ECP), an iron-regulated outer membrane protein (TROMP), and astructural protein of Pasteurellosis; an OMP and a flagellar protein ofVibrosis anguillarum and V. ordalii; a flagellar protein, an OMPprotein,aroA, and purA of Edwardsiellosis ictaluri and E. tarda; andsurface antigen of Ichthyophthirius; and a structural and regulatoryprotein of Cytophaga columnari; and a structural and regulatory proteinof Rickettsia.

[0107] Polypeptides of a parasitic pathogen include but are not limitedto the surface antigens of Ichthyophthirius.

[0108] An “allergen” refers to a substance (antigen) that can induce anallergic or asthmatic response in a susceptible subject. The list ofallergens is enormous and can include pollens, insect venoms, animaldander dust, fungal spores and drugs (e.g. penicillin). Examples ofnatural, animal and plant allergens include but are not limited toproteins specific to the following genuses: Canine (Canis familiaris);Dermatophagoides (e.g. Dermalophagoides farinae); Felis (Felisdomesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Loliumperenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica);Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinoasa);Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa);Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago lanceolata);Parietaria (e.g. Parietaria officinalis orParietaria judaica); Blattella(e.g. Blattella germanica); Apis (e.g. Apis multiflorum); Cupressus(e.g. Cupressus sempervirens, Cupressus arizonica and Cupressusmacrocarpa); Juniperus (e.g. Juniperus sabinoides, Juniperus virginiana,Juniperus communis and Juniperus ashei); Thuya (e.g. Thuya orientalis);Chamaecyparis (e.g. Chamaecyparis obtusa); Periplaneta (e.g. Periplanetaamericana); Agropyron (e.g. Agropyron repens); Secale (e.g. Secalecereale); Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylisglomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis orPoa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus lanatus);Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum (e.g.Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum (e.g.Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum (e.g.Paspalum notatum); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g.Bromus inermis).

[0109] In some aspects of the invention the antigen is a polypeptide.Minor modifications of the primary amino acid sequences of polypeptideantigens may also result in a polypeptide which has substantiallyequivalent antigenic activity as compared to the unmodified counterpartpolypeptide. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. All of the polypeptides produced bythese modifications are included herein as long as antigenicity stillexists. The polypeptide may be, for example, a viral polypeptide. Onenon-limiting example of an antigen useful according to the invention isthe hepatitis B surface antigen. Experiments using this antigen aredescribed in the Examples below.

[0110] The term “substantially purified” as used herein refers to apolypeptide which is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.One skilled in the art can purify viral or bacterial polypeptides usingstandard techniques for protein purification. The substantially purepolypeptide will often yield a single major band on a non-reducingpolyacrylamide gel. In the case of partially glycosylated polypeptidesor those that have several start codons, there may be several bands on anon-reducing polyacrylamide gel, but these will form a distinctivepattern for that polypeptide. The purity of the viral or bacterialpolypeptide can also be determined by amino-terminal amino acid sequenceanalysis.

[0111] The invention also utilizes polynucleotides encoding theantigenic polypeptides. It is envisioned that the antigen may bedelivered to the subject in a nucleic acid molecule which encodes forthe antigen such that the antigen must be expressed in vivo. The nucleicacid encoding the antigen is operatively linked to a gene expressionsequence which directs the expression of the antigen nucleic acid withina eukaryotic cell. The “gene expression sequence” is any regulatorynucleotide sequence, such as a promoter sequence or promoter-enhancercombination, which facilitates the efficient transcription andtranslation of the antigen nucleic acid to which it is operativelylinked. The gene expression sequence may, for example, be a mammalian orviral promoter, such as a constitutive or inducible promoter.Constitutive mammalian promoters include, but are not limited to, thepromoters for the following genes: hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, $-actinpromoter, muscle creatine kinase promoter, human elongation factorpromoter and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the simian virus (e.g., SV40), papilloma virus,adenovirus, human immunodeficiency virus (HIV), rous sarcoma virus,cytomegalovirus (CMV), Rous sarcoma virus (RSV), hepatitis B virus(HBV), the long terminal repeats (LTR) of Moloney leukemia virus andother retroviruses, and the thymidine kinase promoter of herpes simplexvirus. Other constitutive promoters are known to those of ordinary skillin the art. The promoters useful as gene expression sequences of theinvention also include inducible promoters. Inducible promoters areexpressed in the presence of an inducing agent. For example, themetallothionein promoter is induced to promote transcription andtranslation in the presence of certain metal ions. Other induciblepromoters are known to those of ordinary skill in the art.

[0112] In general, the gene expression sequence shall include, asnecessary, 5′ non-transcribing and 5′ non-translating sequences involvedwith the initiation of transcription and translation, respectively, suchas a TATA box, capping sequence, CAAT sequence, and the like.Especially, such 5′ non-transcribing sequences will include a promoterregion which includes a promoter sequence for transcriptional control ofthe operably joined antigen nucleic acid. The gene expression sequencesoptionally include enhancer sequences or upstream activator sequences asdesired.

[0113] The antigen nucleic acid is operatively linked to the geneexpression sequence. As used herein, the antigen nucleic acid sequenceand the gene expression sequence are said to be “operably linked” whenthey are covalently linked in such a way as to place the expression ortranscription and/or translation of the antigen coding sequence underthe influence or control of the gene expression sequence. Two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ gene expression sequence results in the transcription of theantigen sequence and if the nature of the linkage between the two DNAsequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the antigen sequence, or (3) interfere withthe ability of the corresponding RNA transcript to be translated into aprotein. Thus, a gene expression sequence would be operably linked to anantigen nucleic acid sequence if the gene expression sequence werecapable of effecting transcription of that antigen nucleic acid sequencesuch that the resulting transcript is translated into the desiredprotein or polypeptide.

[0114] The antigen nucleic acid sequence may encode a protein,polypeptide, peptide, or peptide mimic of a polysaccharide. It may alsoencode more than one antigenic component as a fusion construct. Morethan one antigen-encoding sequence may be included in the same plasmidvector and these may be linked to the same or different gene expressionsequences.

[0115] The antigen nucleic acid of the invention may be delivered to theimmune system alone or in association with a vector. In its broadestsense, a “vector” is any vehicle capable of facilitating the transfer ofthe antigen nucleic acid to the cells of the immune system andpreferably APCs so that the antigen can be expressed and presented onthe surface of an APC. Preferably, the vector transports the nucleicacid to the immune cells with reduced degradation relative to the extentof degradation that would result in the absence of the vector. Thevector optionally includes the above-described gene expression sequenceto enhance expression of the antigen nucleic acid in APCs. In general,the vectors useful in the invention include, but are not limited to,plasmids, phagemids, viruses, other vehicles derived from viral orbacterial sources that have been manipulated by the insertion orincorporation of the antigen nucleic acid sequences. Viral vectors are apreferred type of vector and include, but are not limited to nucleicacid sequences from the following viruses: retrovirus, such as moloneymurine leukemia virus, harvey murine sarcoma virus, murine mammary tumorvirus, and rouse sarcoma virus; adenovirus, adeno-associated virus;SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papillomaviruses; herpes virus; vaccinia virus; polio virus; and RNA virus suchas a retrovirus. One can readily employ other vectors not named butknown to the art.

[0116] Preferred viral vectors are based on non-cytopathic eukaryoticviruses in which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses, the life cycle ofwhich involves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. Most useful are thoseretroviruses that are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high-efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell lined withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., “Gene Transfer and Expression, A Laboratory Manual”, W. H.Freeman C. O., New York (1990) and Murry, E. J. Ed. “Methods inMolecular Biology”, vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

[0117] Preferred virus for certain applications are the adeno-virus andadeno-associated virus which are double-stranded DNA viruses that havealready been approved for human use in gene therapy and immunotherapytrials. The adeno-associated virus can be engineered to bereplication-deficient and is capable of infecting a wide range of celltypes and species. It further has advantages such as, heat and lipidsolvent stability; high transduction frequencies in cells of diverselineages, including hemopoietic cells; and lack of superinfectioninhibition thus allowing multiple series of transductions. Reportedly,the adeno-associated virus can integrate into human cellular DNA in asite-specific manner, thereby minimizing the possibility of insertionalmutagenesis and variability of inserted gene expression characteristicof retroviral infection. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion.

[0118] Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well-known to those of skill inthe art. See e.g., Sanbrook et al., “Molecular Cloning: A LaboratoryManual”, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been used as DNA vaccines fordelivering antigen-encoding genes to cells in vivo. They areparticularly advantageous for this because they do not have the samesafety concerns as with many of the viral vectors. These plasmids,however, having a promoter compatible with the host cell, can express apeptide from a gene operatively encoded within the plasmid. Somecommonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, andpBlueScript. Other plasmids are well-known to those of ordinary skill inthe art. Additionally, plasmids may be custom designed using restrictionenzymes and ligation reactions to remove and add specific fragments ofDNA. Plasmids such as those used for DNA vaccines may be delivered by avariety of parenteral, mucosal and topical routes. For example theplasmid DNA can be injected by intramuscular, intradermal, subcutaneousor other routes. It may also be administered by intranasal sprays ordrops, rectal suppository and orally. It may also be administered intothe epidermis or a mucosal surface using a gene-gun. The plasmids may begiven in an aqueous solution, dried onto gold particles or inassociation with another DNA delivery system including but not limitedto liposomes, dendrimers, cochleate and microencapsulation.

[0119] It has recently been discovered that gene carrying plasmids canbe delivered to the immune system using bacteria. Modified forms ofbacteria such as Salmonella can be transfected with the plasmid and usedas delivery vehicles. The bacterial delivery vehicles can beadministered to a host subject orally or by other administration means.The bacteria deliver the plasmid to immune cells, e.g. dendritic cells,probably by passing through the gut barrier. High levels of immuneprotection have been established using this methodology.

[0120] In other aspects the invention includes a method for immunizingan infant by administering to an infant an antigen and anoligonucleotide containing at least one unmethylated CpG dinucleotide inan effective amount for inducing cell mediated immunity in the infant.In some embodiments the infant is also administered at least onenon-nucleic acid adjuvant, as described above. Cell mediated immunity,as used herein, refers to an immune response which involves an antigenspecific T cell reaction. The presence of cell mediated immunity can bedetermined directly by the induction of Th1 cytokines (e.g., IFN-,IL-12) and antigen-specific cytotoxic T-cell lymphocytes (CTL). Thepresence of cell mediated immunity is also indicated indirectly by theisotype of antigen-specific antibodies that are induced (e.g.IgG2a>>IgG1 in mice). Thus, if Th1 cytokines or CTL or TH1-likeantibodies are induced, cell mediated immunity is induced according tothe invention. As discussed above, Th1 cytokines include but are notlimited to IL-12 and IFN-γ.

[0121] Neonates (newborn) and infants (which include humans three monthsof age and referred to hereinafter as infants) born in HBV endemic areasrequire particularly rapid induction of strong HBV-specific immunityowing to the high rate of chronicity resulting from infection at a youngage. Without immunoprophylaxis, 70-90% of infants born to motherspositive for both HBsAg and the “e” antigen (HBeAg) become infected andalmost all of these become chronic carriers (Stevens et al., 1987). Evenwhen vaccinated with a four dose regime of the HBV subunit vaccinecommencing on the day of birth, 20% of such infants became chronicallyinfected and this was reduced to only 15% if they were also givenHBV-specific immunoglobulin (Chen et al., 1996). HBV chronicity resultsin 10-15% of individuals infected as adolescents or adults, but 90-95%for those infected (either vertically or horizontally) as infants. CpGoligonucleotides may be used, according to the invention, to reduce thisfurther owing to a more rapid appearance and higher titers of anti-HBsantibodies and the induction of HBV-specific CTL, which could help clearvirus from the liver of babies infected in utero, and which likelyaccount for most of the failures with infant vaccination.

[0122] The invention further provides a method of modulating the levelof a cytokine. The term “modulate” envisions the suppression ofexpression of a particular cytokine when lower levels are desired, oraugmentation of the expression of a particular cytokine when higherlevels are desired. Modulation of a particular cytokine can occurlocally or systemically. CpG oligonucleotides can directly activatemacrophages and dendritic cells to secrete cytokines. No directactivation of proliferation or cytokine secretion by highly purified Tcells has been found, although they are induced to secrete cytokines bycytokines secreted from macrophages and may be costimulated through theT cell Receptor. Cytokine profiles determine T cell regulatory andeffector functions in immune responses. In general, Th1-type cytokinesare induced, thus the immunostimulatory nucleic acids promote a Th1 typeantigen-specific immune response including cytotoxic T-cells.

[0123] Cytokines also play a role in directing the T cell response.Helper (CD4⁺) T cells orchestrate the immune response of mammals throughproduction of soluble factors that act on other immune system cells,including B and other T cells. Most mature CD4⁺ T helper cells expressone of two cytokine profiles: Th1 or Th2. Th1 cells secrete IL-2, IL-3,IFN, GM-CSF and high levels of TNF-α. Th2 cells express IL-3, IL-4,IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-α. The Th1subset promotes both cell-mediated immunity, and humoral immunity thatis characterized by immunoglobulin class switching to IgG_(2a) in mice.Th1 responses may also be associated with delayed-type hypersensitivityand autoimmune disease. The Th2 subset induces primarily humoralimmunity and induce class switching to IgG₁ and IgE. The antibodyisotypes associated with Th1 responses generally have good neutralizingand opsonizing capabilities whereas those associated with Th2 responsesare associated more with allergic responses.

[0124] Several factors have been shown to influence commitment to Th1 orTh2 profiles. The best characterized regulators are cytokines. IL-12 andIFN-γ are positive Th1 and negative Th2 regulators. IL-12 promotes IFN-γproduction, and IFN-γ provides positive feedback for IL-12. IL-4 andIL-10 appear to be required for the establishment of the Th2 cytokineprofile and to down-regulate Th1 cytokine production; the effects ofIL-4 are in some cases dominant over those of IL-12. IL-13 was shown toinhibit expression of inflammatory cytokines, including IL-12 and TNF-αby LPS-induced monocytes, in a way similar to IL-4. The IL-12 p40homodimer binds to the IL-12 receptor and may antagonizes IL-12biological activity; thus it blocks the pro-Th1 effects of IL-12 in someanimals.

[0125] In other aspects the invention includes a method of inducing aTh1 immune response in a subject by administering to the subject acombination of adjuvants in an effective amount for inducing a Th1immune response. The combination of adjuvants includes at least oneoligonucleotide containing at least one unmethylated CpG dinucleotideand at least one non-nucleic acid adjuvant. It was not previously knownthat when CpG was combined with a non-nucleic acid adjuvant, asdescribed above, that the combination would produce an immune responsewith a Th1 profile to an extent that the individual adjuvants could notproduce alone. Preferably the extent of the Th profile produced by thecombination of adjuvants is synergistic. Another aspect of the inventionis to induce a Th response by using CPG with a non-nucleic acid adjuvantthat by itself induces a Th2 response.

[0126] As described above a Th2 profile is characterized by productionof IL-4 and IL-10. Non-nucleic acid adjuvants that induce Th2 or weakTh1 responses include but are not limited to alum, saponins,oil-in-water and other emulsion formulations and SB-As4. Adjuvants thatinduce Th1 responses include but are not limited to MPL, MDP, ISCOMS,IL-12, IFN-γ, and SB-AS2. When the CpG oligonucleotide is administeredwith a non-nucleic acid adjuvant the combination of adjuvants causes acommitment to a Th1 profile, that neither the adjuvant nor the CpGoligonucleotide is capable of producing on its own. Furthermore, if thenon-nucleic acid adjuvant on its own induces a Th2 response, theaddition of CpG oligonucleotide can overcome this Th2 bias and induce aTh1 response that may be even more Th1-like than with CpG alone.

[0127] The combination of adjuvants may be administered simultaneouslyor sequentially. When the adjuvants are administered simultaneously theycan be administered in the same or separate formulations, and in thelatter case at the same or separate sites, but are administered at thesame time. The adjuvants are administered sequentially, when theadministration of the at least two adjuvants is temporally separated.The separation in time between the administration of the two adjuvantsmay be a matter of minutes or it may be longer. The separation in timeis less than 14 days, and more preferably less than 7 days, and mostpreferably less than 1 day. The separation in time may also be with oneadjuvant at prime and one at boost, or one at prime and the combinationat boost, or the combination at prime and one at boost.

[0128] For use in the instant invention, the nucleic acids can besynthesized de novo using any of a number of procedures well known inthe art. For example, the b-cyanoethyl phosphoramidite method (Beaucage,S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981); nucleosideH-phosphonate method (Garegg et al., Tet. Let. 27:4051-4054, 1986;Froehler et al., Nucl. Acid. Res. 14:5399-5407, 1986,; Garegg et al.,Tet. Let. 27:4055-4058, 1986, Gaffney et al., Tet. Let. 29:2619-2622,1988). These chemistries can be performed by a variety of automatedoligonucleotide synthesizers available in the market. Alternatively, CpGdinucleotides can be produced on a large scale in plasmids, (seeSambrook, T., et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor laboratory Press, New York, 1989) which after beingadministered to a subject are degraded into oligonucleotides. Suchplasmids may also encode other genes to be expressed such as anantigen-encoding gene in the case of a DNA vaccine. Oligonucleotides canbe prepared from existing nucleic acid sequences (e.g., genomic or cDNA)using known techniques, such as those employing restriction enzymes,exonucleases or endonucleases.

[0129] For use in vivo, nucleic acids are preferably relativelyresistant to degradation (e.g., via endo-and exo-nucleases). Secondarystructures, such as stem loops, can stabilize nucleic acids againstdegradation. Alternatively, nucleic acid stabilization can beaccomplished via phosphate backbone modifications. A preferredstabilized nucleic acid has at least a partial phosphorothioate modifiedbackbone. Phosphorothioates may be synthesized using automatedtechniques employing either phosphoramidate or H-phosphonatechemistries. Aryl-and alkyl-phosphonates can be made, e.g., as describedin U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which thecharged oxygen moiety is alkylated as described in U.S. Pat. No.5,023,243 and European Patent No. 092,574) can be prepared by automatedsolid phase synthesis using commercially available reagents. Methods formaking other DNA backbone modifications and substitutions have beendescribed (Uhlmaim, E. and Peyman, A., Chem. Rev. 90:544, 1990;Goodchild, J., Bioconjugate Chem. 1:165, 1990).

[0130] For administration in vivo, nucleic acids may be associated witha molecule that results in higher affinity binding to target cell (e.g.,B-cell, monocytic cell and natural killer (NK) cell) surfaces and/orincreased cellular uptake by target cells to form a “nucleic aciddelivery complex.” Nucleic acids can be ionically or covalentlyassociated with appropriate molecules using techniques which are wellknown in the art. A variety of coupling or cross-linking agents can beused, e.g., protein A, carbodiimide, andN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Nucleic acids canalternatively be encapsulated in liposomes or virosomes using well-knowntechniques.

[0131] Nucleic acids containing an appropriate unmethylated CpG can beeffective in any mammal, preferably a human. Different nucleic acidscontaining an unmethylated CpG can cause optimal immune stimulationdepending on the mammalian species. Thus an oligonucleotide causingoptimal stimulation in humans may not cause optimal stimulation in amouse and vice versa. One of skill in the art can identify the optimaloligonucleotides useful for a particular mammalian species of interestusing routine assays described herein and/or known in the art.

[0132] The CpG ODN of the invention stimulate cytokine production (e.g.,IL-6, IL-12, IFN-γ, TNF-α and GM-CSF) and B-cell proliferation in PBMC'staken from a subject such as a human. Specific, but nonlimiting examplesof such sequences include those presented in Table 1 below: TABLE 1sequences GCTAGACGTTAGCGT; (SEQ ID NO:1) GCTAGATGTTAGCGT; (SEQ ID NO:2)GCTAGACGTTAGCGT; (SEQ ID NO:3) GCTAGACGTTAGCGT; (SEQ ID NO:4)GCATGACGTTGAGCT; (SEQ ID NO:5) ATGGAAGGTCCAGCGTTCTC; (SEQ ID NO:6)ATCGACTCTCGAGCGTTCTC; (SEQ ID NO:7) ATCGACTCTCGAGCGTTCTC; (SEQ ID NO:8)ATCGACTCTCGAGCGTTCTC; (SEQ ID NO:9) ATGGAAGGTCCAACGTTCTC; (SEQ ID NO:10)GAGAACGCTGGACCTTCCAT; (SEQ ID NO:11) GAGAACGCTCGACCTTCCAT; (SEQ IDNO:12) GAGAACGCTCGACCTTCGAT; (SEQ ID NO:13) GAGAACGCTGGACCTTCCAT; (SEQID NO:14) GAGAACGATGGACCTTCCAT; (SEQ ID NO:15) GAGAACGCTCCAGCACTGAT;(SEQ ID NO:16) TCCATGTCGGTCCTGATGCT; (SEQ ID NO:17)TCCATGTCGGTCCTGATGCT; (SEQ ID NO:18) TCCATGACGTTCCTGATGCT; (SEQ IDNO:19) TCCATGTCGGTCCTGCTGAT; (SEQ ID NO:20) TCAACGTT; (SEQ ID NO:21)TCAGCGCT; (SEQ ID NO:22) TCATCGAT; (SEQ ID NO:23) TCTTCGAA; (SEQ IDNO:24) CAACGTT; (SEQ ID NO:25) CCAACGTT; (SEQ ID NO:26) AACGTTCT; (SEQID NO:27) TCAACGTC; (SEQ ID NO:28) ATGGACTCTCCAGCGTTCTC; (SEQ ID NO:29)ATGGAAGGTCCAACGTTCTC; (SEQ ID NO:30) ATCGACTCTCGAGCGTTCTC; (SEQ IDNO:31) ATGGAGGCTCCATCGTTCTC; (SEQ ID NO:32) ATCGACTCTCGAGCGTTCTC; (SEQID NO:33) ATCGACTCTCGAGCGTTCTC; (SEQ ID NO:34) TCCATGTCGGTCCTGATGCT;(SEQ ID NO:35) TCCATGCCGGTCCTGATGCT; (SEQ ID NO:36)TCCATGGCGGTCCTGATGCT; (SEQ ID NO:37) TCCATGACGGTCCTGATGCT; (SEQ IDNO:38) TCCATGTCGATCCTGATGCT; (SEQ ID NO:39) TCCATGTCGCTCCTGATGCT; (SEQID NO:40) TCCATGTCGTCCCTGATGCT; (SEQ ID NO:41) TCCATGACGTGCCTGATGCT;(SEQ ID NO:42) TCCATAACGTTCCTGATGCT; (SEQ ID NO:43)TCCATGACGTCCCTGATGCT; (SEQ ID NO:44) TCCATCACGTGCCTGATGCT; (SEQ IDNO:45) GGGGTCAACGTTGACGGGG; (SEQ ID NO:46) GGGGTCAGTCGTGACGGGG; (SEQ IDNO:47) GCTAGACGTTAGTGT; (SEQ ID NO:48) TCCATGTCGTTCCTGATGCT; (SEQ IDNO:49) ACCATGGACGATCTGTTTCCCCTC; (SEQ ID NO:50) TCTCCCAGCGTGCGCCAT; (SEQID NO:51) ACCATGGACGAACTGTTTCCCCTC; (SEQ ID NO:52)ACCATGGACGAGCTGTTTCCCCTC; (SEQ ID NO:53) ACCATGGACGACCTGTTTCCCCTC; (SEQID NO:54) ACCATGGACGTACTGTTTCCCCTC; (SEQ ID NO:55)ACCATGGACGGTCTGTTTCCCCTC; (SEQ ID NO:56) ACCATGGACGTTCTGTTTCCCCTC; (SEQID NO:57) CACGTTGAGGGGCAT; (SEQ ID NO:58) TCAGCGTGCGCC; (SEQ ID NO:59)ATGACGTTCCTGACGTT; (SEQ ID NO:60) TCTCCCAGCGGGCGCAT; (SEQ ID NO:61)TCCATGTCGTTCCTGTCGTT; (SEQ ID NO:62) TCCATAGCGTTCCTAGCGTT; (SEQ IDNO:63) TCGTCGCTGTCTCCCCTTCTT; (SEQ ID NO:64) TCCTGACGTTCCTGACGTT; (SEQID NO:65) TCCTGTCGTTCCTGTCGTT; (SEQ ID NO:66) TCCATGTCGTTTTTGTCGTT; (SEQID NO:67) TCCTGTCGTTCCTTGTCGTT; (SEQ ID NO:68) TCCTTGTCGTTCCTGTCGTT;(SEQ ID NO:69) TCCTGTCGTTTTTTGTCGTT; (SEQ ID NO:70)TCGTCGCTGTCTGCCCTTCTT; (SEQ ID NO:71) TCGTCGCTGTTGTCGTTTCTT; (SEQ IDNO:72) TCCATGCGTGCGTGCGTTTT; (SEQ ID NO:73) TCCATGCGTTGCGTTGCGTT; (SEQID NO:74) TCCACGACGTTTTCGACGTT; (SEQ ID NO:75) TCGTCGTTGTCGTTGTCGTT;(SEQ ID NO:76) TCGTCGTTTTGTCGTTTTGTCGTT; (SEQ ID NO:77)TCGTCGTTGTCGTTTTGTCGTT; (SEQ ID NO:78) GCGTGCGTTGTCGTTGTCGTT; (SEQ IDNO:79) TGTCGTTTGTCGTTTGTCGTT; (SEQ ID NO:80) TGTCGTTGTCGTTGTCGTTGTCGTT;(SEQ ID NO:81) TGTCGTTGTCGTTGTCGTT; (SEQ ID NO:82) TCGTCGTCGTCGTT; (SEQID NO:83) TGTCGTTGTCGTT; (SEQ ID NO:84) TCCATAGCGTTCCTAGCGTT; (SEQ IDNO:85) TCCATGACGTTCCTGACGTT; (SEQ ID NO:86) GTCGYT; (SEQ ID NO:87)TGTCGYT; (SEQ ID NO:88) AGCTATGACGTTCCAAGG; (SEQ ID NO:89)TCCATGACGTTCCTGACGTT; (SEQ ID NO:90) ATCGACTCTCGAACGTTCTC; (SEQ IDNO:91) TCCATGTCGGTCCTGACGCA; (SEQ ID NO:92) TCTTCGAT; (SEQ ID NO:93)ATAGGAGGTCCAACGTTCTC; (SEQ ID NO:94) GTCGTT (SEQ ID NO:95) GTCGTC (SEQID NO:96) TGTCGTT (SEQ ID NO:97) TGTCGCT (SEQ ID NO:98)

[0133] Preferred CpG ODN can effect at least about 500 pg/ml of TNF-, 15pg/ml IFN- , 70 pg/ml of GM-CSF 275 pg/ml of IL-6, 200 pg/ml IL-12,depending on the therapeutic indication. These cytokines can be measuredby assays well known in the art. The oligonucleotides listed above orother preferred CpG ODN can effect at least about 10%, more preferablyat least about 15% and most preferably at least about 20% YAC-1 cellspecific lysis or at least about 30%, more preferably at least about35%, and most preferably at least about 40% 2C11 cell specific lysis, inassays well known in the art.

[0134] The term “effective amount” of a CpG oligonucleotide refers tothe amount necessary or sufficient to realize a desired biologic effect.For example, an effective amount of an oligonucleotide containing atleast one unmethylated CpG and a non-nucleic acid adjuvant for treatingan infectious disorder is that amount necessary to cause the developmentof an antigen specific immune response upon exposure to the microbe,thus causing a reduction in the amount of microbe within the subject andpreferably to the eradication of the microbe. The effective amount forany particular application can vary depending on such factors as thedisease or condition being treated, the particular CpG oligonucleotidebeing administered (e.g. the number of unmethylated CpG motifs or theirlocation in the nucleic acid), the size of the subject, or the severityof the disease or condition. One of ordinary skill in the art canempirically determine the effective amount of a particular adjuvant andantigen without necessitating undue experimentation.

[0135] The formulations of the invention are administered inpharmaceutically acceptable solutions, which may routinely containpharmaceutically acceptable concentrations of salt, buffering agents,preservatives, compatible carriers, adjuvants, and optionally othertherapeutic ingredients.

[0136] For use in therapy, an effective amount of the adjuvantcombination can be administered to a subject by any mode allowing theoligonucleotide to be taken up by the appropriate target cells.“Administering” the pharmaceutical composition of the present inventionmay be accomplished by any means known to the skilled artisan. Preferredroutes of administration include but are not limited to oral,transdermal (e.g. via a patch), parenteral injection (subcutaneous,intradermal, intravenous, parenteral, intraperitoneal, intrathecal,etc.), or mucosal intranasal, intratracheal, inhalation, andintrarectal, intravaginal etc). An injection may be in a bolus or acontinuous infusion.

[0137] For example the pharmaceutical compositions according to theinvention are often administered by intramuscular or intradermalinjection, or other parenteral means, or by biolistic “gene-gun”application to the epidermis. They may also be administered byintranasal application, inhalation, topically, intravenously, orally, oras implants, and even rectal or vaginal use is possible. Suitable liquidor solid pharmaceutical preparation forms are, for example, aqueous orsaline solutions for injection or inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of present methods for drug delivery, see Langer, Science249:1527-1533, 1990, which is incorporated herein by reference.

[0138] The pharmaceutical compositions are preferably prepared andadministered in dose units. Liquid dose units are vials or ampoules forinjection or other parenteral administration. Solid dose units aretablets, capsules and suppositories. For treatment of a patient,depending on activity of the compound, manner of administration, purposeof the immunization (i.e., prophylactic or therapeutic), nature andseverity of the disorder, age and body weight of the patient, differentdoses may be necessary. The administration of a given dose can becarried out both by single administration in the form of an individualdose unit or else several smaller dose units. Multiple administration ofdoses at specific intervals of weeks or months apart is usual forboosting the antigen-specific responses.

[0139] The adjuvants and antigens may be administered per se (neat) orin the form of a pharmaceutically acceptable salt. When used in medicinethe salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof. Such salts include,but are not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, such salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group.

[0140] Suitable buffering agents include: acetic acid and a salt (1-2%w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5%w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitablepreservatives include benzalkonium chloride (0.003-0.03% w/v);chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal(0.004-0.02% w/v).

[0141] The pharmaceutical compositions of the invention contain aneffective amount of a combination of adjuvants and antigens optionallyincluded in a pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” means one or more compatible solidor liquid filler, dilutants or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being comingled with the compounds of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

[0142] Compositions suitable for parenteral administration convenientlycomprise sterile aqueous preparations, which can be isotonic with theblood of the recipient. Among the acceptable vehicles and solvents arewater, Ringer's solution, phosphate buffered saline and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed mineral or non-mineral oil may be employed including syntheticmono-ordi-glycerides. In addition, fatty acids such as oleic acid finduse in the preparation of injectables. Carrier formulations suitable forsubcutaneous, intramuscular, intraperitoneal, intravenous, etc.administrations may be found in Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa.

[0143] The adjuvants or antigens useful in the invention may bedelivered in mixtures of more than two adjuvants or antigens. A mixturemay consist of several adjuvants in addition to the synergisticcombination of adjuvants or several antigens.

[0144] A variety of administration routes are available. The particularmode selected will depend, of course, upon the particular adjuvants orantigen selected, the age and general health status of the subject, theparticular condition being treated and the dosage required fortherapeutic efficacy. The methods of this invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of an immuneresponse without causing clinically unacceptable adverse effects.Preferred modes of administration are discussed above.

[0145] The compositions may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing the compounds intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the compounds into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

[0146] Other delivery systems can include time-release, delayed releaseor sustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. They include polymerbase systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-di-and tri-glycerides; hydrogelrelease systems; sylastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which an agent of the invention iscontained in a form within a matrix such as those described in U.S. Pat.Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems inwhich an active component permeates at a controlled rate from a polymersuch as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.In addition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

[0147] The present invention is further illustrated by the followingExamples, which in no way should be construed as further limiting. Theentire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference.

EXAMPLES

[0148] The use of CpG ODN as an adjuvant alone or in combination withother adjuvants was evaluated. The hepatitis B virus surface antigen(HBsAg) given as a recombinant protein or expressed in vivo from a DNAvaccine was used as an exemplary model system in the Examples set forthbelow.

[0149] Materials and Methods

[0150] Animals

[0151] Experiments on adult mice were carried out using female BALB/cmice (Charles River, Montreal, QC) at 6-8 weeks of age.

[0152] Newborn mice were obtained through breeding male and femaleBALB/c mice (Charles River) in the Loeb animal facility (Loeb HealthResearch Institute, The Ottawa Hospital, Ottawa, ON). Pregnant femaleswere monitored daily to ensure accurate recording of the date of birth.Both male and female neonates were used for immunization.

[0153] Cynomolgus monkeys (1.5-3 kg) were housed at the Primate ResearchCenter, Bogor, Indonesia.

[0154] Orantutans (5-20 kg) were housed at Wanariset Station for theOrangutan Reintroduction Program of the Indonesian government,Balikpapan, Kalimantan.

[0155] HBsAg Subunit Vaccination of Mice

[0156] The subunit vaccine consisted of HBsAg (ay subtype) which hadbeen produced as a recombinant protein in yeast cells (Medix Biotech#ABH0905). This was diluted in saline for use without adjuvant. HBsAgwas also formulated with alum and/or CpG ODN as adjuvant. HBsAg proteinwas mixed with aluminum hydroxide (Alhydrogel 85, [Al₂O₃], SuperfosBiosector, Vedbaek, Denmark) in the same ratio of 25 mg Al³⁺ per mgprotein as used in the commercial vaccines (i.e., 2.5 l 2% Al₂O₃ per μgHBsAg). The protein and alum were mixed with a vortex and then left onice for at least 30 minutes prior to use to allow the protein to adsorbonto the Al₂O₃. This solution was mixed again immediately prior toinjection by drawing up into the syringe 3-5 times.

[0157] For groups treated with CpG ODN, an appropriate volume ofsynthetic oligodeoxynucleotide (ODN #1826) of the sequenceTCCATGACGTTCCTGACGTT (SEQ ID NO. 86) synthesized with a phosphorothioatebackbone (Oligos Etc. & Oligo Therapeutics, Wilsonville, Oreg.) wasadded alone or with alum to HBsAg on the day of injection. Adult micereceived a single intramuscular (IM) injection into the left tibialisanterior (TA) muscle of 1 or 2 ug HBsAg, without or with adjuvant (alumand/or CpG ODN), in 50 l vehicle. When CpG DNA was added, each animalreceived a total of 1, 10, 100 or 500 μg ODN. Newborn mice wereimmunized within 24 hours of birth or 7 days after birth by bilateralinjection of a total of 1 μg HBsAg into the posterior thigh muscles(2×10 l@0.05 mg/ml). All injections were carried out with a 0.3 mlinsulin syringe which has a fused 29G needle (Becton Dickenson, FranklinLakes, N.J.). For injection of adults, the needle was fitted with acollar of polyethylene (PE) tubing to limit penetration of the needle toabout 3 mm. All intramuscular injections were carried out through theskin (shaved for adults) and under general anesthesia (Halothane,Halocarbon Laboratories, River Edge, N.J.).

[0158] HBsAg Subunit Vaccination of Monkeys

[0159] Monkeys were immunized by IM injection into the anterior thighmuscle of Engerix-B® (SmithKline Beecham Biologicals, Rixensart, BE)which comprises HBsAg (ay subtype, 20 μg/ml) adsorbed to alum (25 mgAl3+/mg HbsAg). Each monkey received an injection of 0.5 ml containing10 μg HbsAg. For some monkeys, 500 μg CpG ODN 1968(TCGTCGCTGTTGTCGTTTCTT) (SEQ ID NO 72) was added to the vaccineformulation.

[0160] HBsAg Subunit Vaccination of Orangutans

[0161] Orangutans were immunized by IM injection into the anterior thighmuscle of HBsAg *ay subtype, 20 μg/ml) combined with alum (25 mg Al3+/mgHBsAg), combined with CpG. CpG ODN 2006 (TCGTCGTTTTGTCGTTTTGTCGTT) (SEQID NO 77) was added to the vaccine formulation. Each orangutan receivedan injection of 1.0 ml containing 20 μg HBsAg with alum (500 μg), CpGoligonucleotide (1 mg) or both adjuvants.

[0162] Experimental Groups

[0163] Comparison of CpG ODN and Non-Nucleic Acid Adjuvants with HBsAgSubunit Vaccine

[0164] Twelve groups of adult BALB/c mice (n=10) were injected with 1 μgHBsAg (i) alone, (ii) mixed with alum, (iii, iv, v, vi, vii) mixed with0.1, 1, 10, 100 or 500 μg CpG ODN, or (viii, ix, x, xi, xii) mixed withboth alum and 0.1, 1, 10, 100 or 500 μg CpG ODN. These mice were bled at1, 2, 4 and 8 weeks after immunization and the plasma was assayed foranti-HBs. At the end of the study the mice were killed and their spleensremoved for assay of CTL activity.

[0165] Other groups of mice (n=5) were immunized with HBsAg (1 μg)alone, with alum (25 μg Al3+), with one of several different CpG andnon-CpG control oligonucleotides of different backbones (10 μg), or withboth alum and an oligonucleotide.

[0166] Other groups of mice (n=5) were immunized as above (except onlythe 10 μg dose of CpG ODN was used) and boosted with the identical or adifferent formulation at 8 weeks, then spleens were removed 2 weekslater for evaluation of CTL activity.

[0167] Other groups of mice were immunized with HBsAg (1 μg) and one ofthe following non-nucleic acid adjuvants alone or in combination withCpG ODN (10 μg): monophosphoryl lipid A (MPL, 50 μg, Ribi); Freund'sComplete Adjuvant (CFA; 1:1 v/v); Freund's Incomplete Adjuvant (IFA; 1:1v/v).

[0168] Immunization of Neonates with Subunit or DNA Vaccine

[0169] Groups of newborn and young BALB/c mice (n=10) aged <24 hours, 3,7 or 14 days were injected with (i, ii, iii) a total of 1 μg HBsAg withalum, with CpG ODN 1826 (10 μg) or with both alum and CpG ODN, or with(iv) an HBsAg-expressing DNA vaccine (1-μg pCMV-S). Plasma was obtainedat 4, 8, 12 and 16 weeks for assay of anti-HBs as total IgG and IgGsubtypes (IgG1 and IgG2a). At the end of the study the mice were killedand their spleens removed for assay of CTL activity.

[0170] Immunization of Cynomolgus Monkeys With HBsAg and Alum orAlum+CpG ODN

[0171] Two groups of juvenile Cynomolgus monkeys (n=5) were immunized at0 and 10 weeks with 0.5 ml Engerix-B (HBsAg at 20 mg/ml adsorbed toalum, 25 mg Al3+/mg HBsAg) to which had been added saline (0.1 ml) orCpG ODN 2006 (500 μg in 0.1 ml, SEQ ID #77). Monkeys were bled at 2, 8,10, 12 and 14 weeks and plasma was evaluated for anti-HBs titers(mIU/ml).

[0172] Immunization of Orangutans With HBsAg and Alum or CpG ODN orAlum+CpG ODN

[0173] Three groups of juvenile orangutans were immunized IM at 0 and 4weeks with 1 ml of vaccine containing HBsAg (10 μg) plus (i) alum (25 mgAl3+/mg HBsAg)(n=13), (ii) CpG ODN 2006 (SEQ#77) (m=24) or (iii) alumplus CpG ODN (n=14). Animals were bled at 4.8 and 12 weeks and plasmawas evaluated for anti-HBs titers (mIU/ml).

[0174] Evaluation of Humoral Response to HbsAg

[0175] Mice: Heparinized blood was collected by retrobulbar puncture oflightly anaesthetized mice as described elsewhere (Michel et al., 1995).Plasma was recovered by centrifugation (7 min@13,000 rpm). Antibodiesspecific to HBsAg in plasma were detected and quantified by end-pointdilution ELISA assay (in triplicate) on individual samples. Ten-foldserial dilutions of plasma were first added to 96-well microtiter plateswith a solid phase consisting of plasma-derived HBsAg particles (100l/well of HBsAg ay subtype at 1 g/ml, coated overnight at RT) andincubated for 1 hr at 37C. The bound antibodies were then detected byincubation for 1 hr at 37C with HRP-conjugated goat anti-mouse IgG, IgM,IgG1 or IgG2a (1:4000 in PBS-Tween, 10% FCS; 100 l/well, SouthernBiotechnology Inc., Birmingham, Ala.), followed by incubation with OPDsolution (100 l/well, Sigma, St. Louis, Mo.) for 30 minutes at RT in thedark. The reaction was stopped by the addition of sulfuric acid (50 l of4N H₂SO₄). End-point titers were defined as the highest plasma dilutionthat resulted in an absorbance value (OD 450) two times greater thanthat of non-immune plasma with a cut-off value of 0.05. Anti-HBs titerswere expressed as group means of individual animal values, which werethemselves the average of triplicate assays.

[0176] Primates: Monkeys and orangutans were bled by antecubital venouspuncture into heparinized tubes and plasma was recovered bycentrifugation (7 min@13,000 rpm). Plasma was then evaluated foranti-HBs titers using a commercial kit (Monolisa anti-HBs,Pasteur-Sanofi) and expressed in milli-International Units permilliliter (mIU/ml) by comparison with standards defined by the WorldHealth Organization. A titer of 10 mIU/ml is considered sufficient toconfer protection to humans and great apes against infection by HBV.

[0177] Evaluation of Cytotoxic T Cell Response to HBsAg in Mice

[0178] Cytotoxic T-lymphocyte (CTL) activity was determined usingsplenocytes taken from mice 4 or 8 weeks post-prime or post-boost. Inbrief, single cell suspensions were prepared and suspended in tissueculture medium (RPMI 1640, 10% FBS, Life Technologies, Grand Island,N.Y., supplemented with 5×10⁻⁵ M-mercaptoethanol andpenicillinstreptomycin solution, 1000 U/ml, 1 mg/ml final concentrationsrespectively, Sigma, and 3% E1-4 supernatant as a source of IL-2).Splenocytes (3×10⁷) were co-cultured for 5 days (37° C., 5% CO2) with1×10⁶ syngenic HBsAg-expressing stimulator cells (P815S) or controltarget cells (P815) in round bottom 96-well culture plates (37° C., 5%CO2, 4 hr). Supernatant (100 μl) was removed for radiation (gamma)counting. Spontaneous release was determined by incubating target cellswithout effector cells and total release by addition of 100 μl 2 N HClto the target cells. The percent lysis was calculated as [(experimentalrelease—spontaneous release)/(total release—spontaneous release)]×100.The percent specific lysis was calculated as % lysis with P815S-% lysiswith P815 cells. CTL activity for responding mice [% specific lysis>10]were expressed as means±SEM of individual animal values, which werethemselves the average of triplicate assays.

Example 1 Synergy of CpG ODN With Alum as Adjuvant for HBV SubunitVaccine in Mice

[0179] A. Strength and Kinetics of Humoral Response

[0180] Immunization of BALB/c mice with HBsAg alone elicited only lowtiters of anti-HBs (<100) by 4 weeks. These titers were about 10-foldhigher with the addition of alum as adjuvant, 60-fold higher with CpGODN and more than 500-fold higher with both alum and CpG ODN. At latertime points, the highest peak titers were with HBsAg/alum/CpG, thesecond highest with HBsAg/CpG, then HBsAg/alum (FIG. 1).

[0181] Similar synergistic results for antibody responses were obtainedwhen immunization against HBsAg was carried out in neonatal and veryyoung mice, in which the immune system is immature. In mice immunized at3 days of age, where the immune system is even less mature than anewborn human, 10% and 0% of mice seroconverted with alum and CpG ODNalone respectively, but 75% serocoinverted when CpG ODN and alum wereused together. In 7 day old mice, which have an immune system similar inmaturity to that of a newborn human, seroconversion for alum, CpG or thecombination was 11%, 22% and 100% respectively (FIG. 8). Furthermore, inthese 7 day old mice, antibody titers were up to 80-fold higher with thecombined adjuvants than with either adjuvant alone (FIG. 9).

[0182] When used alone or combined with alum, there is a dose-responsefor CpG ODN with the best results being obtained with an intermediatedose (10 μg) and no further or only relatively small gains with higherdoses (up to 500 μg) when used alone or combined with alum respectively(FIG. 2).

[0183] When a large panel of ODN is compared for adjuvant activity itcan be seen that CPG ODN with a nuclease-resistant phosphorothioatebackbone have the best adjuvant effects (FIG. 3). There was very littleor no adjuvant activity of non-CpG control ODN with a phosphorothioatebackbone, or of CpG ODN with a chimeric or phosphodiester backbone.However, for those phosphorothioate CpG ODN that did not have adjuvanteffect, all exhibited a synergistic effect with alum. In general,antibody titers with combined alum and CpG ODN were 10 to 100-foldhigher than with CpG ODN and/or 100 to 1000-fold higher than with alumalone (FIG. 3).

[0184] B. Strength of Cylotoxic T-Lymphocyte Response

[0185] CTL were very weak with HBsAg and no adjuvant, and werecompletely lost with the addition of alum. CTL were augmented equallywith both CpG ODN as with combined alum and CpG ODN (FIG. 1). A synergyfor CTL responses could be seen with prime-boost strategies, in thatpriming with CpG ODN and boosting with alum gave better CTL than primingand boosting with CpG alone (FIG. 4) (Note: use of alum alone completelyabrogates the CTL response).

[0186] A synergistic action of CpG ODN and alum on CTL was very evidentwith immunization of young (7 day old) mice. In this case, neither alumnor CpG ODN used alone induced significant levels of HBsAg-specific CTL,but when used together there wre very strong CTL were observed (FIG. 9).

[0187] Thus, CpG ODN is superior to alum for both humoral andcell-mediated responses, when each is used alone as adjuvant with theHBsAg subunit vaccine in mice. When used together, there is a synergy ofaction such that antibody and CTL activity are stronger than when eitheradjuvant is used alone. These results indicate that CpG ODN could beused to replace alum in vaccine formulations, which could be desirableto avoid associated side-effects due to local irritation in the muscle,or for certain live-attenuated or multivalent vaccines where it is notpossible to use alum because chemical interactions interfere with theefficacy of the vaccine. This should not occur with CpG ODN. Of evengreater interest is the strong synergistic response when CpG ODN andalum are used together as adjuvants. This could allow better immuneresponses with lower or fewer doses of antigen. There is a fairly flatdose response to CpG ODN whether or not alum is present, indicating thata wide range of CpG ODN could be useful to adjuvant vaccines in humans.

Example 2 Synergy of CpG ODN With other Non-Nucleic Acid Adjuvants forHBV Subunit Vaccine in Mice

[0188] As discussed above, CpG ODN alone gave 8-fold higher antibodytiters than alum, the only adjuvant currently licensed for human use. Italso produces superior results to monphosphoryl lipid A (MPL, RibiPharmaceuticals, Middleton, Wis.), a new adjuvant that is currently inhuman clinical trials even when administered in a dose of five timesless than that of MPL. There was, as discussed above, a strong synergywith CpG ODN and alum, but in contrast no such synergy was seen with MPLand alum. Owing to the strong synergistic effect of alum and CpG ODN,this combination of adjuvants is even better than Freund's completeadjuvant (FCA) for inducing antibodies in mice (FIG. 5) Freund's, whichis considered the gold standard adjuvant for animal models is much tootoxic to use in humans.

[0189] The synergy seen with CpG ODN and alum, was also seen with CpGODN combined with other adjuvants. When used alone, CpG ODN and Freund'sincomplete adjuvant (FIA, a type of mineral oil) induced similarantibody titers, but when used together the anti-HBs titers were morethan 50-fold higher than with either adjuvant alone. Indeed, thecombination of CpG ODN and FIA was even better than FCA (FIG. 6).

[0190] Similarly, CpG ODN and MPL alone gave equally high antibodytiters, but when used together the titers were about 4-times higher thanwith either adjuvant alone (FIG. 7). While the synergistic response withCpG and MPL was not as marked with respect to overall antibody titers,it was very pronounced with respect to the Th1-bias of these antibodies(see below).

Example 3 Dominance and Synergy of CpG ODN With Alum for Induction of aTh1-Type Immune Response Including CTL

[0191] Immunization with either HBsAg alone or with alum induces apredominantly Th2-type humoral response with virtually no IgG2aantibodies, which are induced in response to Th1-type cytokines such asIL-12 and IFN-. Rather, almost all (>99%) antibodies were of the IgG1isotype IgG2a:IgG1=0.01. CpG ODN induces significantly more IgG2aantibodies, such that they made up at least 50% of the total IgG (IgG(IgG2a:IgG1=1.4). The combination of alum and CpG ODN induce an equallystrong Th1 response as CpG ODN alone (IgG2a:IgG1=1.0), despite theextremely strong Th2-bias of alum (FIG. 5). Similarly CTL responses withCpG ODN plus alum were as strong as those with CpG ODN alone, despitethe fact that the Th2-bias of alum resulted in a complete loss of CTLwhen alum was used alone (FIG. 1).

[0192] The strong Th1 bias with CpG is even more evident in neonatal andyoung mice, which are known to naturally have a strong Th2-bias to theirimmune system. In this case, neither alum nor CpG ODN on their owninduced detectable IgG2a, indicating a very poor or absent Th1 response.Remarkably, when used together, CpG ODN and alum induced high levels ofIg G2a antibodies, which were now the predominant form of IgG (FIG. 10).Similarly, neither CpG ODN or alum induced significant levels of CTL inyoung mice, yet when used together there was a strong CTL response, thatwas even stronger than obtained with a DNA vaccine (FIG. 9).

[0193] The strength of the Th1 influence of CpG ODN is seen not only byits ability to dominate over the Th2 effect of alum when they areco-administered, but also to induce Th1 responses in animals previouslyprimed for a Th2 response with alum. Immunization with HBsAg using alumas an adjuvant completely abrogates the CTL response owing to the strongTh2 bias of alum (FIGS. 1 and 4). However, in mice using alum at primeand CpG at boost, good CTL were induced, indicating the possibility ofCpG to overcome a previously established Th2 response (FIG. 4).

[0194] Aluminum hydroxide (alum) is currently the only adjuvant approvedfor human use. An important disadvantage of alum is that it induces aTh2- rather than a Th1-type immune response, and this may interfere withinduction of CTL. Indeed, in mice immunized with recombinant HBsAg, theaddition of alum selectively blocked activation of CD8⁺ CTL (Schirmbecket al., 1994). Although not essential for protective immunity againstHBV, CTL may nevertheless play an important role. For example, a lack ofHBV-specific CTL is thought to contribute to the chronic carrier state.In contrast, one of the primary advantages of CpG DNA over alum as anadjuvant is the Th1-bias of the responses and thus the possibility toinduce CTL. A striking finding from the present study is that CpG cancompletely counteract the Th2-bias of alum when the two adjuvants aredelivered together, and in the case of immunization in early life, thecombination can even give a more Th1 response than CpG ODN alone. Thiscould allow one to capitalize on the strong synergistic action of thetwo adjuvants on the humoral response while still allowing CTL inadults, and to induce a stronger Th1 response in infants.

[0195] The use of alum has been linked to Th2-type diseases. The muchhigher prevalence of asthma (another Th2-type disease) in more highlydeveloped nations may be linked to the high hygiene level and rapidtreatment of childhood infections (Cookson and Moffatt, 1997). Earlyexposure to bacterial DNA (and immunostimulatory CpG motifs) pushes theimmune system away from Th2- and towards a Th1-type response and thismay account for the lower incidence of asthma in less developedcountries, where there is a much higher frequency of upper respiratoryinfections during childhood. Addition of CpG ODN as adjuvant to allpediatric vaccines could re-establish a Th1-type response therebyreducing the incidence of asthma.

Example 4 Synergy of CpG ODN With Other Adjuvants for Induction of aTh1-Type Immune Responses

[0196] The synergistic effect of CpG ODN on Th1 responses was also seenusing other adjuvants. IFA on its own induces a very strong Th2-typeresponse with virtually no IgG2a antibodies (IgG2a:IgG1=0.002) and CpGODN on its own induces a moderate Th1 response (IgG2a:IgG1=1.4), buttogether the response was very strongly Th1 (IgG2a:IgG1=24.0). It isnotable that this is even more Th1 than the response induced by CFA(ratio=0.5) (FIG. 6).

[0197] Similarly, CpG and MPL on their own are moderately Th1(IgG2a:IgG1 ratios at 4 weeks are 1.4 and 1.9 respectively), buttogether are very strongly Th1 with a large predominance of IgG2aantibodies (ratio=83.3)(FIG. 7).

Example 5 CpG ODN as Synergistic Adjuvant in Cynomolgus Monkeys

[0198] CpG ODN, in combination with alum, also acts as a potent adjuvantto augment anti-HBs responses in Cynomolgus monkeys. Compared toresponses obtained with the commercial HBV vaccine that contains alum,monkeys immunized with the commercial vaccine plus CpG ODN attainedtiters 50-times higher after prime and 10-times higher after boost (FIG.14).

Example 6 CpG ODN as Synergistic Adjuvant to HBsAg in HyporesponderOrangutans

[0199] The orangutans, a great ape very closely related to man, can benaturally infected with HBV in a manner similar to humans.Unfortunately, orangutans are hyporesponsive to the commercial HBVvaccine that contains alum as an adjuvant. Compared to humans, where 13%and 56% of vaccinated individuals seroconvert by 4 weeks after first andsecond doses respectively (Yano and Tashiro, 1988), only 0% and 15% ofvaccinated orangutans have seroconverted by the same times. With theaddition of 1 mg CpG ODN, this becomes 43% and 100% respectively. Asynergistic response is seen even in these hyporesponders, becauseantibody levels and seroconversion rates are better with CpG ODN plusalum than with either adjuvant alone (FIG. 12).

[0200] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by examplesprovided, since the examples are intended as a single illustration ofone aspect of the invention and other functionally equivalentembodiments are within the scope of the invention. Various modificationsof the invention in addition to those shown and described herein willbecome apparent to those skilled in the art from the foregoingdescription and fall within the scope of the appended claims. Theadvantages and objects of the invention are not necessarily encompassedby each embodiment of the invention.

REFERENCES

[0201] 1 Chen D. S. et al. (1996). Cancer Causes & Control 7:305-311.

[0202] 2. Cookson, W. O. C. M. & Moffatt, M. F. (1997). Science 275:41-42.

[0203] 3. Cowdery, J. S. et al. (1996). J. Immunol. 156: 4570.

[0204] 4. Davis, H. L. et al. (1993). Human Molec. Genet. 2: 1847-1851.

[0205] 5. Davis, H. L. et al. (1995). Human Gene Ther. 6: 1447-1456.

[0206] 6. Davis, H. L. et al. (1996). Proc. Natl. Acad. Sci. USA 93:7213-7218.

[0207] 7. Davis, H. L. et al. (1996). Vaccine 14: 910-915.

[0208] 8. Davis, H. L. et al. (1997). Gene Ther. (in press).

[0209] 9. Davis, H. L. & Brazolot Millan, C. L. (1997). Blood CellBiochem. (in press)

[0210] 10. Donnelly, J. J. et al. (1996). Life Sciences. 60: 163-172.

[0211] 11. Dubois, M. -F. et al. (1980). Proc. Natl. Acad. Sci. USA 77:4549-4553.

[0212] 12. Ellis, R. W. (Ed.) (1993). Hepatitis B Vaccines in ClinicalPractise. New York: Marcel-Dekker.

[0213] 13. Halpern, M. D. et al. (1996). Cell. Immunol. 167: 72.

[0214] 14. Klinman, D. M. et al. (1996). Proc. Natl. Acad. Sci. USA 93:2879-2883.

[0215] 15. Krieg, A. M. et al. (1995). Nature 374: 546-549.

[0216] 16. Kruskall, M. S. et al. (1992). J. Exp. Med. 175: 495-502.

[0217] 17. Lee C. Y. et al., (1989). J. Am. Med. Assoc. SEA

[0218] 18. Li et al.,(1994). J. Gen. Virol. 75: 3673-3677.

[0219] 19. Mancini, M. et al. (1996). Proc. Natl. Acad. Sci. USA 93:12496-12501.

[0220] 20. Michel, M. -L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:7708-7712.

[0221] 21. Michel, M. -L. et al. (1995). Proc. Natl. Acad. Sci. USA 92:5307-5311.

[0222] 22. Milich, D. R. (1988). Immunol. Today 9: 380-386.

[0223] 23. Niederau et al. (1996). New Eng. J. Med. 334: 1422-1427.

[0224] 24. Pol, S. et al. (1993). C. R. Acad. Sci. (Paris) 316: 688-691.

[0225] 25. Rehermann, B. et al. (1996). Nature Med. 2: 1104-1108.

[0226] 26. Sato, Y., et al. (1996). Science 273:352-354.

[0227] 27. Schirmbeck, R. et al. (1994). J. Immunol. 152: 1110-1119.

[0228] 28. Stevens, C. E. et al. (1987). J. Am. Med. Assoc. 257:2612-2616.

[0229] 29. Vogel, F. R. & Sarver, N. (1995). Clin. Microbiol. Rev. 8:406-410.

[0230] 30. Yano, M and Tashiro, A. (1988) In: Viral Hepatitis and LiverDisease, Ed: A. J. Zuckerman; Alan R. i, New York, pp 1038-1042.

[0231] All references, patents and patent publications that are recitedin this application are incorporated in their entirety herein byreference.

We claim:
 1. A method for treating a subject infected with hepatitisvirus comprising: administering to a subject infected with hepatitisvirus, wherein the subject is a non-responder or hypo-responder, anoligonucleotide, having a sequence including at least the followingformula: 5′X₁X₂CGX₃X₄3′ wherein the oligonucleotide includes at least 8nucleotides wherein c is unmethylated and wherein X₁X₂ and X₃X₄ arenucleotides, in an effective amount to produce an immune responseagainst the hepatitis virus.
 2. The method of claim 1, furthercomprising administering a non-nucleic acid adjuvant.
 3. The method ofclaim 1, further comprising administering an antigen.
 4. The method ofclaim 2, wherein the non-nucleic acid adjuvant is an immune stimulatingadjuvant.
 5. The method of claim 4, wherein the immune stimulatingadjuvant is selected from the group consisting of saponins, PCPPpolymer, derivatives of lipopolysaccharides, MPL, MDP, t-MDP, OM-174 andLeishmania elongation factor.
 6. The method of claim 2, wherein thenon-nucleic acid adjuvant is an adjuvant that creates a depo effect andstimulates the immune system.
 7. The method of claim 6, wherein theadjuvant that creates a depo effect and stimulates the immune system isselected from the group consisting of ISCOMS, SB-AS2, SB-AS4, non-ionicblock copolymers, and SAF (Syntex Adjuvant Formulation).
 8. The methodof claim 1, wherein the 5′X₁X₂CGX₃ X₄ 3′ sequence is a non-palindromicsequence.
 9. The method of claim 1, wherein the nucleic acid includes abackbone having at least one phosphate backbone modification.
 10. Themethod of claim 9, wherein the phosphate backbone modification is amodification of the first two internucleotide linkages of the 5′ end ofthe nucleic acid.
 11. The method of claim 9, wherein the phosphatebackbone modification is a modification of the last two internucleotidelinkages of the 3′ end of the nucleic acid.
 12. The method of claim 1,wherein the oligonucleotide is 8 to 100 nucleotides in legnth.
 13. Themethod of claim 9, wherein the phosphate backbone modification is aphosphorothioate or phosphorodithioate modification.
 14. The method ofclaim 1, wherein X₁X₂ are nucleotides selected from the group consistingof: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; andX₃X₄ are nucleotides selected from the group consisting of: TpT, CpT,ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA.
 15. The method ofclaim 1, wherein X₁X₂ are selected from the group consisting of GpA andGpT and X₃X₄ are TpT.
 16. The method of claim 1, wherein X₁X₂ are bothpurines and X₃X₄ are both pyrimidines.
 17. The method of claim 1,wherein X₂ is a T and X₃ is a pyrimidine.
 18. The method of claim 1,wherein the oligonucleotide is 8 to 40 nucleotides in length.
 19. Themethod of claim 1, wherein the oligonucleotide is a syntheticoligonucleotide.
 20. The method of claim 1, wherein the hepatitis virusis hepatitis B.
 21. The method of claim 1, wherein the hepatitis virusis hepatitis C.